WO2013050998A1 - Protéine cd247 utilisée comme biomarqueur en vue de l'évaluation de l'effet de médicaments chimiothérapeutiques et biologiques - Google Patents

Protéine cd247 utilisée comme biomarqueur en vue de l'évaluation de l'effet de médicaments chimiothérapeutiques et biologiques Download PDF

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WO2013050998A1
WO2013050998A1 PCT/IL2012/050393 IL2012050393W WO2013050998A1 WO 2013050998 A1 WO2013050998 A1 WO 2013050998A1 IL 2012050393 W IL2012050393 W IL 2012050393W WO 2013050998 A1 WO2013050998 A1 WO 2013050998A1
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treatment
expression
determining
sample
cells
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PCT/IL2012/050393
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Michal Baniyash
Julia KANTERMAN
Moshe SADE-FELDMAN
Eliran ISH-SHALOM
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Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.
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Priority to US14/348,621 priority Critical patent/US10955415B2/en
Priority to EP12784738.2A priority patent/EP2834640B1/fr
Publication of WO2013050998A1 publication Critical patent/WO2013050998A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/7051—T-cell receptor (TcR)-CD3 complex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/44—Multiple drug resistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates to personalized medicine. More specifically, the invention provides methods, kits and compositions for assessing the efficacy and selecting an appropriate therapy such as chemo therapeutic, biological therapy or combined therapy for treating a subject suffering from a pathologic disorder that leads to a chronic- inflammatory condition.
  • an appropriate therapy such as chemo therapeutic, biological therapy or combined therapy for treating a subject suffering from a pathologic disorder that leads to a chronic- inflammatory condition.
  • Cancers of cancer that are based on the immune system have been used in clinical trials for years but were not shown yet as a successful strategy for an overall extension of survival [1-4].
  • the main strategy in the treatment of cancer is chemotherapy, sometimes accompanied with targeted or general immunotherapy [1 , 4- 7].
  • Chemotherapy protocols focus on the destruction of cancer cells, for example, by preventing cancer cells from further multiplying or by inducing cell death, thereby eliminating cancer cells from the body.
  • Chemotherapeutic drugs systemically affect the whole body by being transmitted through the bloodstream and are thus able to eliminate cancer cells at sites that are distant from the site of the original tumor [4, 8]. Unfortunately, during the course of such a process healthy cells are also affected, especially those that are naturally rapidly dividing [7]. While chemotherapy is advantageous in patients at initial stages of developing tumors, advanced cancer patients usually poorly benefit from such treatments, mostly without an option for a cure [9-11]. Thus, although chemotherapy is a very common and sometimes efficacious treatment, there is an urgent need for a less harmful and more targeted strategy, due to its toxic effects.
  • immunotherapy which is a more controlled therapeutic strategy [1-2, 4-7, 12].
  • Immunotherapy constitutes of a tumor specific biologic therapy that mimics or uses certain parts of the immune system to destruct or eliminate cancer cells.
  • the concept of immunotherapy relies on the natural defense system of the body, which naturally protects against a variety of diseases [5, 7, and 13].
  • Immunotherapeutic strategies that are currently used include boosting the patient's own immune system, for example, by vaccination against cancer targets, by cytokine treatment that modulate the activity of the immune system of the patient, or by administrating to a patient engineered versions of normal components of the immune system (e.g. adoptive immune cell transfer, administering antitumor, anti-receptor, or anti-cytokine antibodies, administering soluble cytokine receptors, etc.). Due to the high specificity for cancer cells, the toxicity of immunotherapy is rather low, and in some cases, supports and complements chemotherapy treatment [1, 4-7].
  • the generated tumor microenvironment exhibits features that support tumor growth, for example, by developing local chronic inflammatory response, which later turns systemic [14-18]. While chronic inflammation can directly support tumor growth by secreted factors and enriched populations of unique cells, which may lead to an enhanced tumor cell survival or proliferation and angiogenesis, chronic inflammation may also indirectly support tumor growth by inducing immunosuppression [14, 17 and 19], all leading to a failure of the patient's immune system in defeating the tumor. Therefore, in many cases, the success rates of the above-mentioned therapies are very limited, ensuing in malignant prospers and metastasis. Today there is an urgent need for biomarkers that are required to measure the host's immune status and inflammatory state prior to a given therapy, as well as during its course and following the therapy.
  • the inventors have previously established a mouse model system mimicking chronic inflammation induced immunosuppressive conditions WO 2005/025310. This publication further disclosed the use of CD247 as a marker for the immunological status of a patient suffering from a chronic inflammatory condition. Another previous publication of the inventors, WO 2009/125408, have demonstrated the use of CD247 as a diagnostic and prognostic marker for monitoring the immune status of patients suffering from chronic inflammatory conditions, specifically, diabetes.
  • the invention relates to a method for determining the efficacy of a treatment with a therapeutic agent on a subject suffering from a chronic inflammatory condition, more specifically, the method of the invention provides determining whether a subject suffering from a pathologic condition that may lead to a chronic inflammation, would respond, by exhibiting a beneficial response to a treatment with a therapeutic agent.
  • the therapeutic agent used for treating this subject may be at least one chemo therapeutic agent, at least one biological therapy agent, at least one immunotherapeutic agent or any combination thereof.
  • the method of the invention may comprise the steps of: In a first step (a), determining the level of expression of T cell antigen receptor (TCR) ⁇ chain (CD247) in at least one biological sample of said subject, to obtain an expression value. It should be noted that the examined sample must be obtained after initiation of the treatment.
  • TCR T cell antigen receptor
  • CD247 T cell antigen receptor
  • the next step (b) involves determining if the expression value obtained in step (a) is any one of, positive, negative or equal to a predetermined standard expression value or to an expression value of CD247 in a control sample. Determination of a positive or negative expression value may be performed by comparing the expression value obtained in step (a) to a predetermined standard expression value or to an expression value of CD247 in a control sample, and calculating the differences between said expression values.
  • a positive expression value of CD247 in the tested sample indicates that the subject responds to the treatment and moreover, exhibits a beneficial response to the treatment. More specifically, a positive expression value indicates that the examined subject belongs to a pre-established population associated with a beneficial response to the specific treatment. In contrast, a negative expression value indicates that the examined subject does not respond to said treatment and more specifically, does not exhibit a beneficial response to the treatment.
  • the method of the invention provides determination of the efficacy of a specific treatment on a specific subject that suffers from a chronic inflammatory condition.
  • the invention relates to a composition comprising:
  • detecting molecules of (a) and (b) may be attached to a solid support.
  • a third aspect of the invention relates to a kit comprising:
  • FIG. 1A-1C Irinotecan (CPT11) increases a chronic inflammatory response
  • FIG. 1A Graphic representation of the accumulation of Grl + Macl + MDSCs at day +2 after BCG treatment in the absence or in the presence of CPT11 treatment administered i.p (left) or i.v. (right) in the spleen.
  • Fig. IB Graphic representation of the accumulation of Grl + Macl + MDSCs at day +2 after BCG treatment in the absence or in the presence of CPT11 treatment administered i.p (left) or i.v. (right) in peripheral blood cells (PBLs).
  • sp. spleen
  • cont. control
  • PBL peripheral blood
  • FIG. 1A-2E The expression of CD247 in BCG mice treated with CPT11
  • FIG. 2A Graphic representation of splenocytes (left) and PBLs (right) obtained from mice untreated, inflamed (BCG-treated) or inflamed that were subjected to i.p. or i.v. irinotecan treatment that were fixed, permeabilized and double stained for total expression of CD247 ( ⁇ chain).
  • Fig. 2C Splenocytes from untreated, inflamed, and inflamed-treated i.v. with irinotecan were labeled with CFSE and activated with anti-CD3 and anti-CD28 antibodies or left non- activated. The proliferative response was assessed by monitoring cell divisions of gated CFSE-labeled Thyl .2+ (CD90+) cells. Data was calculated by percent of proliferating cells compared to steady state of non-activated of each group.
  • Fig. 2D In- vivo cytotoxicity assay of CFSElabeled allogeneic (CFSElow) and syngeneic (CFSEhigh) splenic-derived cell clearance by NK cells was assessed 18-24h following administration. A representative data of NK killing activity from three independent experiments of CFSE cell clearance within the spleens and PBLs from mice untreated, inflamed, and inflamed-treated i.v. with irinotecan, is shown.
  • sp. spleen
  • cont. control
  • PBL peripheral blood
  • Fig. 3A Splenocytes from mice untreated, inflamed, and inflamed treated i.v. with irinotecan, were prepared for intracellular nitric oxide (NO) detection, stained with non- flouroscent diamino fluorescein -2 Diacetate (DAF-2DA) and double stained for Grl+ CD1 lc+ (MDSCs). NO production, gated on MDSCs, was measured as shown by MFI.
  • Fig. 3B Detection of highly reactive oxygen species (hROS) secretion was performed by loading cells with APF and incubated with or without stimulator, LPS, at 37oC and after washing, double stained for Grl+ CDl lc+ (MDSCs).
  • hROS highly reactive oxygen species
  • FIG. 5A-5C Cyclophosphamide enhances chronic inflammation-dependent immunosuppression
  • Fig. 5C Accumulation of MDSCs at day +2 after BCG-induced chronic inflammation in peripheral blood cells (PBLs).
  • PBLs peripheral blood cells
  • Data from three independent experiments (n 4).
  • FIG. 6A-6D Cyclophosphamide enhances T- and NK-cell dysfunction under chronic inflammation
  • Fig. 6B CD3s expression levels are shown by mean fluorescence intensity (MFI). Statistical analyses using t test indicated significant differences at 95% CI. Means and SEM are shown. Fig. 6C, Fig. 6D.
  • Fig. 7A Splenocytes from mice untreated, inflamed, and inflamed treated i.p. with cyclophosphamide were prepared for intracellular nitric oxide (NO) detection, stained with no n-flouro scent diamino fluorescein -2 Diacetate (DAF-2DA) and double stained for Grl+ CDl lc+ (MDSCs). NO production, gated on MDSCs, was measured as shown by MFI.
  • NO nitric oxide
  • DAF-2DA n-flouro scent diamino fluorescein -2 Diacetate
  • MDSCs double stained for Grl+ CDl lc+
  • Figure 8A-8C Doxorubicin (Rubex) reduces the immunosuppressive effect of a chronic inflammatory response
  • Fig. 8A and 8B Accumulation of MDSCs at day +2 after the course of BCG treatment induces chronic inflammation in the spleen (Fig. 8A) and peripheral blood cells (PBLs) (Fig. 8B).
  • Treatment i.p. of inflamed mice with doxorubicin reduced MDSC levels in the spleen (Fig. 8A) and PBLs (Fig. 8B).
  • Figure 9A-9D Doxorubicin restores CD247 expression in T- and NK-cells under chronic inflammation
  • Splenocytes (Fig. 9A, 9C) and PBLs (Fig. 9B, 9D) from mice non-inflamed, inflamed, and inflamed treated i.p. with doxorubicin were fixed, permeabilized and double stained for total expression of the CD247 (Fig. 9A, 9B) or CD3_ (Fig. 9C, 9D), as shown by mean fluorescence intensity (MFI).
  • Statistical analyses using t test indicated significant differences at 95% CI. Means and SEM are shown. *, P ⁇ 0.05; **, P ⁇ 0.01 ; ***, P ⁇ 0.001. Abbreviations: sp. (spleen), cont. (control), PBL. (peripheral blood).
  • FIG. 11A-11B Busulfan restores CD247 expression under chronic inflammation
  • FIG. 12A-12C 5-fluorouracil (5-FU) neutralizes chronic inflammation induced immunosuppression
  • FIG. 13A-13E 5-FU restores T- and NK-cell function under chronic inflammation
  • FIG. 13D, 13E In-vivo NK cell cytotoxicity assay assessed by clearance of CFSE labeled allogeneic (CFSElow) and syngeneic (CFSEhigh) splenic-derived cells, 18-24h following their administration.
  • CFSElow CFSE labeled allogeneic
  • CFSEhigh syngeneic
  • FIG. 13D A representative data of NK killing activity from three independent experiments of CFSE cell clearance within the spleens (Fig. 13D) and PBLs (Fig. 13E) from non-inflamed, inflamed, and inflamed mice treated i.p. with 5- FU, is shown.
  • Statistical analyses using t test indicated significant differences at 95% CI. Means and SEM are shown. *, P ⁇ 0.05. Abbreviations: cont. (control), Kil. Rat (killing rate).
  • Fig 14 Administration of 5-FU leads to a restoration of CD247 expression of adoptively transferred T cells
  • Fig 15A-15H Administration of etanercept increases MDCS differentiation and reduces immunosuppression
  • Fig. 15A A schematic presentation of the mouse model used. WT-mice were injected daily with etanercept from 1 day prior to the second BCG injection until 1 day before mice were sacrificed. Etanercept-treated mice were compared with non-inflamed and inflamed WT- and KO-mice.
  • Fig. 15CAccumulation of MDSCs 2 days after the last BCG treatment in the spleen, of etanercept-treated and untreated mice (n 7). *P ⁇ 0.02, **P ⁇ 0.003 (t-test). Data are representative of two independent experiments (error bars, s.d.).
  • Fig. 15CAccumulation of MDSCs 2 days after the last BCG treatment in the spleen, of etanercept-treated and untreated mice (n 7). *P ⁇ 0.02, **P ⁇ 0.003 (t-test). Data are representative of two independent experiments (error bars,
  • FIG. 16A-16D Selected chemotherapeutics and their in-vivo effect on MDSCs
  • FIG. 16A Schematic representation of the mouse model for chronic inflammation.
  • Fig. 16B 16 C.
  • chemotherapy treatments saline, control mice; Rubex, Doxorubicin; Busulfan; 5-FU, 5-fiuorourcil, CPT-11, Irinotecan; CP, Cyclophosphamide
  • Fig. 16B, top spleens (bottom; representative 5-FU and CPT-11 plots)
  • FIG. 16C peripheral blood cells (PBLs) were isolated and MDSCs (Grl + CD1 lb + ) accumulation was revealed by flow cytometry analysis.
  • Graphs represent the percent of MDSCs presented within the spleen and PBLs.
  • FIG. 17A-17F 5-FU and CPT-11 monotherapies display opposite effect on MDSC suppressive activity
  • Fig. 17C Real Time PCR analyses were performed to check the levels of inflammatory cytokines (TNFa, IL-6, IFNy) following administration of 5-FU or CPT11, relative to the expression in inflamed mice with no chemotherapy treatment (set as 1). Statistical analyses using t test indicated significant differences at 95%.
  • Fig. 17F Splenic MDSCs from each group were analyzed for the expression percentage of cleaved caspase-3, revealed by flow cytometry analysis gating on MDSC (Grl+CDl lb + ) populations. Statistical analyses using t test indicated significant differences at 95%. Means and SEM are shown. *, P ⁇ 0.05; **, P ⁇ 0.01. Abbreviations: Inf. (inflamed), Tot. sp. (total spleen), sp (spleen), g. (gated), Norm, (normal).
  • Fig. 18A-18E Splenocytes and PBLs from mice untreated (naive), inflamed, or inflamed and treated with 5-FU or CPT-11 were fixed, permeabilized, and double stained for the expression of CD247 ( ⁇ -chain) (Fig. 18 A, 18B) and CD3s-chain (Fig. 18D, 18E), as shown by mean fluorescence intensity (MFI), measured in gated CD3 + cells.
  • Fig. 18C Splenocytes from mice, naive, inflamed and inflamed treated with 5-FU or CPT-11 , were labeled with CFSE and activated with anti-CD3 and anti-CD28 antibodies or left non-activated.
  • Figure 19A-19C Differential effects of 5-FU and CPT-11 monotherapies on NK- cell activity
  • Figs. 19B, 19C In-vivo cytotoxicity assay of CFSE-labeled allogeneic (CFSE low ) and syngeneic (CFSE 1 " 811 ) splenic-derived cell clearance by NK- cells was assessed 18-24h following administration. Representative data of NK killing activity from three independent experiments of CFSE cell clearance within the (b) spleens and (c) PBLs from naive, inflamed, and inflamed- 5-FU or CPT-11 treated mice, is shown. Abbreviations: Nor. (normal), clear rat. (clearance rate), Inf. (inflamed),
  • Fig. 20A, 20G spleens were isolated and analyzed for MDSC levels by flow cytometry. Statistical analyses using t test indicated significant differences at 95%. Means and SEM are shown. *, P ⁇ 0.05.
  • Fig. 20E, Fig. 20F In-vivo NK cell cytotoxicity assay assessed by clearance of CFSE labeled allogeneic (CFSElow) and syngeneic (CFSEhigh) splenic-derived cells, 18-24h following their administration.
  • CFSElow allogeneic
  • CFSEhigh syngeneic
  • Fig. 20F A representative data of NK killing activity from three independent experiments of CFSE cell clearance within the spleens (Fig. 20F) and PBLs (Fig. 20E) from non-inflamed, inflamed, and inflamed mice treated i.p. with a combination of 5-FU and CPT11 , is shown.
  • Statistical analyses using t test indicated significant differences at 95% CI. Means and SEM are shown.
  • FIG. 21A-21D A mouse model for colorectal cancer (CRC) associated with chronic inflammation and the effect of mono-chemotherapies
  • Figs 21B, 21D. Splenocytes (Fig. 21B) and PBLs (Fig. 21D) from mice naive, CRC-bearing, or CRC- bearing and treated with 5-FU or CPT-11 were fixed, permeabilized, and double stained for the expression of CD247 ( ⁇ -chain). Samples were subjected to FACS analysis. Data from three independent experiments (n 3) is presented. Statistical analyses using t test indicated significant differences at 95%. Means and SEM are shown. **, P ⁇ 0.01. Abbreviations: Norm, (normal).
  • Figure 22A-22D Combined chemotherapies on the immune status of CRC-bearing mice
  • FIG. 23A-23B 5-FU monotherapy and 5-FU with CPTll combined therapy display opposite effect on MDSC suppressive activity
  • FIG. 24A-24H The immunosuppressive environment is detected within the tumor target site
  • FIG. 25A, 25B Colon structure and survival curves of CRC-bearing mice treated with 5fU or 5FU/ CPT11.
  • Monitoring the function of the immune system in patients suffering from a disease or health conditions associated with chronic inflammation may enable inter alia, identifying responders vs. non-responders to immune-based therapies, to evaluate therapeutic efficacies in cases of immune- or chemo-therapy, and also to follow the disease regression or recurrence.
  • Such monitoring is expected to lead to an intelligent selection of the timing, the amount, and of the nature of an agent to be administered for treatment of a disease or health condition associated with a chronic inflammation, such that this agent may be used at the personalized level.
  • the quality of life of such patients could be significantly improved, with the expectation of fewer disease cases such as in cancer declined tumor progression and metastasis and avoiding opportunistic infections.
  • such a monitoring may also reduce the high expenses required for continuous costly patient's examinations, which may be less frequently performed.
  • the Inventors have discovered the biomarker CD247 protein that may be used to fulfill the abovementioned requirements. While the expression of the CD247 molecule is steady during both normal conditions and acute inflammation, it is modulated in the curse of chronic inflammation; down-regulation of the CD247 biomarker correlates with the immune-suppression induced by chronic inflammation [19-21].
  • CD247 may serve to evaluate the host' s immune status.
  • CD247 may be used for predicting the success of given immune-based therapies (e.g. vaccination, antibody-mediated, adoptive cell transfer, etc), for evaluating the beneficial effects of biological therapies [(e.g. etanercept, anti-inflammatory drugs (sildenafil)] and more importantly, for determining the effect of various chemotherapeutic drugs.
  • immune-based therapies e.g. vaccination, antibody-mediated, adoptive cell transfer, etc
  • biological therapies e.g. etanercept, anti-inflammatory drugs (sildenafil)
  • chemotherapeutic agents or drugs affect the immune system of the host. While some chemotherapeutic agents were sown by the Inventors to be beneficial in neutralizing the chronic inflammatory-induced immune-suppression (e.g. 5-fiuorouracil), other chemotherapeutic agents enhanced the immunosuppressive environment, resulting in an increased propensity that the tumor will escape the host's immune response and in an increased risk of metastasis.
  • chemotherapeutic agents were sown by the Inventors to be beneficial in neutralizing the chronic inflammatory-induced immune-suppression (e.g. 5-fiuorouracil)
  • other chemotherapeutic agents enhanced the immunosuppressive environment, resulting in an increased propensity that the tumor will escape the host's immune response and in an increased risk of metastasis.
  • NK cells innate cells
  • T cells adaptive (T cells) immune systems
  • CD247 ⁇ chain
  • SNX9 WO 2012/104836
  • Chronic inflammation-induced immune-suppression was identified to be mediated by myeloid-derived suppressor cells (MDSCs), which are observed during various inflammatory conditions including in tumor-bearing mice, as well as in patients with various types of cancer [21-24].
  • MDSCs myeloid-derived suppressor cells
  • MDSCs represent a heterogeneous population of immature myeloid cells originating in the bone marrow and in the course of chronic inflammation, are highly expanding in peripheral lymphatic organs, imposing their immunosuppressive function and eliminating any anti-tumor immune response [21 , 24- 26].
  • the present invention is also based on the findings that the generated immunosuppressive environment sensed by measuring the expression levels of the biomarker CD247 is harmful to various immune based therapies.
  • Adoptively transferred T and NK cells lose their immune function within 24 hours and thus, the host may be unable to respond to a given vaccination.
  • the host's immune system is recovered, enabling a successful response to immune-based therapy.
  • CD247 may be used as a biomarker for identifying the effect of chemotherapeutic agents or biologic or antiinflammatory drugs on the immune system of the host.
  • Chemotherapeutic agents were found to be either harmful to the immune system, i.e. enhancing the immune- suppression state of the subject by elevating the numbers of MDSCs and the suppressive activity thereof, and resulting in further CD247 down regulation associated with a more pronounced immunosuppression.
  • other chemotherapeutic agents were found to be beneficial to the host, as they decrease the numbers of MDSCs and thus the suppressive activity thereof, ensuing in the recovery of the expression levels of CD247.
  • the present invention is based on the finding that the expression level of CD247 in cells can allocate patients suffering from chronic inflammation and associated immune- suppression.
  • CD247 can be used as a specific and reliable biomarker for selecting a suitable treatment for said patient such that the immune-suppressive environment of the patient will not be further aggravated or cured.
  • the general propose of the present invention is to manipulate the host's inflammatory environment and MDSCs' harmful effects in order to increase the potency of the host's immune response as the anti-tumor immune response in cases of cancer, by taking advantage of known chemotherapy treatments with or without a combination of immune-based or biological therapeutic strategies.
  • the first step was to assess clinically approved chemotherapeutic agents for their mode of function and allocating those that not only affect the tumor but also have the capacity to counteract the tumor associated chronic inflammation-induced immunosuppression.
  • mice have used their previously established mouse model system that mimics the chronic inflammation-induced immunosuppressive conditions, as observed in hosts with developing tumors. These mice were treated with several chemotherapy cell cycle specific and non-specific (alkylating) drugs, and immune-based and biological drugs using therapeutic concentrations approved by the FDA.
  • the present invention show for the first time that CD247 could be used as biomarker for detecting the effect of chemotherapeutic and biologic drugs on the hosts' immune system under developing chronic inflammatory conditions as observed in some cancer patients.
  • CD247 expression level in secondary lymphatic organs, peripheral blood and sites of the growing tumors (biopsies) prior to and/or following treatments the inventors discovered that chemotherapeutic drugs not only act in arresting tumor growth, but also influence dramatically the individual's immune status.
  • the present invention shows a significant suppression of the immune system following treatment with several chemotherapeutic agents, that involves not only a depletion of lymphocytes as was reported previously but also as inducers of extremely high expansion of MDSCs in the periphery, lymphatic organs and sites of growing tumors, resulting in impaired immune function within the host, and neutralization of any therapeutic strategy that is based upon stimulating/enhancing the host's immune response or on donor adoptively-transferred cells.
  • the present invention shows that while some chemotherapy treatments [Doxorubicin (Rubex), Busulfan and 5- fluorouracil (5-FU)] lead to immune recovery from the immunosuppressive state observed in mice with chronic inflammation, surprisingly, other drugs [Irinotecan (CPT11) and Cyclophosphamide (CP)] lead to opposing results; highly increasing peripheral immunosuppression as indicated by changes in several key parameters in the spleen, peripheral blood and specific sites of growing tumors.
  • the biomarker CD247 which senses the individual's immune status, was dramatically down-regulated, indicating an immunosuppressive stage.
  • the inventors show that MDSC levels were increased and their maturation state decreased, MDSC suppressive function was elevated, measured by nitric oxide (NO-) and reactive oxygen species (ROS) secretion, and in-vivo killing activity mediated by NK cells and T cell proliferative capacity were dramatically reduced, all parameters implying an enhanced immunosuppressive environment. Therefore, the vision of the present inventors is that in cases of cancer, treatment must be dually targeted against the tumor cells and the chronic inflammatory microenvironment (that may lead to a systemic chronic inflammatory condition). The first aimed at tumor destruction and the second at breaking the immunosuppressive stage.
  • NO- nitric oxide
  • ROS reactive oxygen species
  • Chemotherapy can weaken immunity by causing a drop in the number of white blood cells, and leading to an increased MDSCs accumulation, resulting in a strong immunosuppression and failure of host's immune system to fight against the residing tumor cells or failure of administered immune-based or biological treatments.
  • the results of the present invention highlight a new concept that CD247 can serve as a tool for detecting whether some chemotherapy drugs have the capacity to neutralize and others enhance immunosuppression. This concept can be crucial when designing cancer treatments aimed at avoiding disease recurrence. Moreover, this could form the basis for considering combinatorial treatment combining chemotherapy with immune-based or biological therapy to induce a global surrounding supporting anti-tumor immune response.
  • the present invention provides as well new data on the use novel biomarkers that could sense the immune status prior to and/or following a given therapy and their use in establishing optimal personalized treatments.
  • the invention relates to a method for determining the efficacy of a treatment with a therapeutic agent on a subject suffering from a pathologic disorder that leads to a chronic inflammatory condition. More specifically, the method of the invention provides and enables determining whether a subject suffering from a chronic inflammatory condition would respond, and specifically, exhibit a beneficial response to a treatment with a therapeutic agent.
  • the therapeutic agent used for treating this subject may be at least one chemotherapeutic agent, at least one immunotherapeutic agent, biologic agent or any combination thereof. More specifically, the method of the invention may comprise the steps of:
  • a first step (a) determining the level of expression of T cell antigen receptor (TCR) ⁇ chain (CD247) in at least one biological sample of said subject, to obtain an expression value.
  • TCR T cell antigen receptor
  • CD247 T cell antigen receptor ⁇ chain
  • the next step (b) involves determining if the expression value obtained in step (a) is any one of, positive, negative or equal to a predetermined standard expression value (that is also referred to herein as a cutoff value) or to an expression value of CD247 in a control sample. Determination of a positive or negative expression value may be performed by comparing the expression value obtained in step (a) to a predetermined standard expression value or to an expression value of CD247 in a control sample. Such a step involves calculating and measuring the difference between the expression values of the examined sample and the cutoff value and determining whether the examined sample can be defined as positive or negative.
  • a predetermined standard expression value that is also referred to herein as a cutoff value
  • comparing denotes any examination of the expression level and/or expression values obtained in the samples of the invention as detailed throughout in order to discover similarities or differences between at least two different samples. It should be noted that comparing according to the present invention encompasses the possibility to use a computer based approach.
  • a positive expression value of CD247 in the tested sample indicates that the subject responds to the treatment and moreover, may exhibit a beneficial response to the treatment.
  • the predetermined standard values are calculated and obtained from populations of subjects suffering from the same chronic inflammatory condition that responded well to the same therapeutic agent, subjects not responding, healthy subjects and untreated subjects.
  • control samples are used instead of, or in addition to predetermined cutoff values, such controls may include subjects suffering from the same chronic inflammatory condition that responded well to the same therapeutic agent, subjects not responding, healthy subjects and untreated subjects.
  • a positive expression value or an equal value when compared to cutoff representing the responder population), reflect up-regulation of CD247 expression, and indicates that the examined subject belongs to a pre-established population associated with a beneficial response to the specific treatment.
  • up-regulation should be considered as up-regulation relatively to the expression prior to the initiation of the treatment.
  • such up-regulation may be expression of CD247 in the range of or similar to the levels of the expression in healthy subjects (age and gender matched) that do not suffer from any chronic inflammatory condition.
  • a negative expression value that is a result of down regulated expression of CD247, indicates that the examined subject does not respond to said treatment and more specifically, does not exhibit a beneficial response to the treatment.
  • down-regulated expression reflects a decrease in the expression of CD247that is below the expression levels in healthy or responder subjects.
  • the method of the invention provides determining the suitability for treatment of a patient suffering from a disease associated with a chronic inflammatory condition a- priori, i.e., before the onset of such treatment, or in most cases, in early stages of the treatment, enabling a personalized treatment.
  • the method of the invention will enable avoiding a treatment that will potentially aggravate the chronic inflammatory condition or the immune-suppressive environment in said patient and the selection of a treatment that will be beneficial to the specific patient.
  • the method of the invention provides determining the suitability and efficacy of treatment of a patient suffering from a disease associated with a chronic inflammatory condition during said treatment, i.e. after the onset thereof, to monitor the effect of said treatment on the patient.
  • the expression value of CD247 decline below a predetermined value or below the expression value in a control sample obtained from the patient prior to the onset of the treatment, or during the treatment, the treatment may be ceased, and alternative treatments may be sought for by the method of the invention, thus avoiding the deleterious effect that may accompany ensuing such treatment on the immune system.
  • the method of the invention provides the use of CD247 as a biomarker for sensing the effect of a therapeutic agent, specifically, chemotherapeutic drug, anti- inflammatory drug, biological drug, on a patient and thereby determining the efficacy of a suggested treatment on a particular patient.
  • a therapeutic agent specifically, chemotherapeutic drug, anti- inflammatory drug, biological drug
  • CD247 also termed CD3 zeta and "T-cell receptor T3 zeta chain” (also known by other human synonyms, including T3Z, CD3H, CD3Q, CD3Z, TCRZ and CD3-ZETA).
  • CD247 together with T-cell receptor alpha/beta and gamma/delta heterodimers, and with CD3- gamma, -delta and -epsilon, forms the T-cell receptor-CD3 complex.
  • the zeta chain plays an important role in coupling antigen recognition to several intracellular signal- transduction pathways. Low expression of the antigen results in impaired immune response.
  • the T-cell antigen receptor is a multisubunit receptor complex specific to T cells subserving both antigen recognition and signal transduction functions.
  • the CD247 [zeta (Q chain] of the TCR is a component of all surface receptor complexes. Sequence analysis of cDNAs encoding human and murine ⁇ revealed that it is a highly conserved protein. In addition to amino acid homology, there is remarkable interspecies conservation in the nucleotide sequence of the 5' and 3' untranslated regions of the ⁇ mRNA. The ⁇ subunit has no sequence similarity to the CD3 chains and the localization of the human ⁇ gene to the centromeric region of chromosome 1 underscores the fact that it is a distinct genetic component of the TCR.
  • CD247 protein refers to the human CD247 protein. More specifically, in humans, several variants of CD247 were reported.
  • the human CD247 protein is as denoted by the sequence herein referred to as SEQ ID NO. 1 (GeneBank accession number J04132.1), encoded by the nucleic acid sequence as denoted by SEQ ID NO. 2.
  • SEQ ID NO. 1 GeneBank accession number J04132.1
  • other variants of CD247 may be also applicable for the present invention.
  • Non limiting examples include the human CD247 proteins as also referred to by the accession number GI: 62898210, GI: 164696323 and GI: 19344013.
  • CD247 sequences are also denoted by the terms “T-cell surface CD3 zeta chain isoform 1 precursor” and “T-cell surface CD3 zeta chain isoform 2 precursor. Still further, the invention refers (specifically in the Examples section) in some particular embodiments to CD247 [Mux musculus] that is a mouse, CD247 protein of GeneBank accession number M19729.1. According to certain embodiments, the amino acid sequence of said mouse CD247 as referred to herein is denoted by the sequence SEQ ID NO. 3, encoded by the nucleic acid sequence as denoted by SEQ ID NO. 4.
  • the method of the invention may comprise a further step of calculating the rate of change in the expression value of CD247 in response to the specific examined treatment.
  • the rate of change in the expression in response to treatment reflects the responsiveness of the examined subject to the particular therapeutic agent.
  • the rate of change in CD247 expression indicates if the treated subject exhibits a beneficial response for such treatment. More specifically, such method may comprise the steps of:
  • the first step (a) involves determining the level of expression of CD247 in at least one biological sample of the examined subject to obtain an expression value. It should be noted that the sample is obtained after the initiation of the examined treatment.
  • the level of expression of CD247 is determined in at least one more biological sample of the same subject to obtain an expression value. It should be noted that the sample may be obtained prior to initiation of said treatment.
  • step (c) the rate of change between the expression value obtained in step (a), and the expression value obtained in step (b) is calculated.
  • step (d) involves determination if the rate of change obtained in step (c) is any one of, positive, negative or equal to a predetermined standard rate of change (cutoff value). More specifically, such standard rate of change is determined prior and after the initiation of treatment with the same therapeutic agent, for population of subjects suffering from the same pathologic disorder defined as "responders". It should be also understood that in certain embodiments, the level of CD247 in a sample after treatment, may also be evaluated as compared to standard value obtained for a population of age and gender matched healthy subject that do not suffer from any pathologic disorder.
  • this step involves determining if the rate of change is positive, negative or equal as compared to the rate of change calculated for expression values in at least one control sample obtained prior and following the specific treatment.
  • Such step involves calculating and measuring the difference between the rate of change in the expression values of the examined sample and the standard rate of change (cutoff value) and determining whether the examined sample can be defined as positive or negative.
  • the standard rate of change (cutoff value) were calculated for populations of patients suffering from the same pathologic disorder (that are not treated), subjects treated with the same therapeutic agent (responders or non-responders) and healthy subjects (age and gender matched). It should be also appreciated that the predetermined standard values (cutoff) may be presented in a standard curve.
  • control samples may include samples from patients suffering from the same pathologic disorder (untreated or treated with the specific therapeutic agent), subjects suffering from the same condition treated with the same therapeutic agent that are responders, samples of non-responders and samples of healthy subjects (age and gender matched).
  • a positive or equal rate of change of CD247 expression value reflects elevation in the expression of CD247 in response to said treatment relative to prior sample and or cut offs as above and therefore indicates that the examined subject responds to the certain treatment and specifically exhibits a beneficial response to the treatment. More particularly, a positive rate of change indicates that the examined subject belongs to a pre-established population associated with a higher probability to exhibit a beneficial response to the specific examined treatment. A negative expression value relative to prior sample and or cut offs as above indicates that the examined subject does not respond and specifically, does not exhibit a beneficial response to the treatment.
  • a negative rate of change reflects reduction of CD247 expression in response to treatment with a specific therapeutic compound revealing a chronic inflammatory state in the patient and thereby indicating that the specific therapeutic agent is not appropriate for said examined patient.
  • the efficacy of the specific treatment in a specific subject is thereby determined.
  • the method of the invention may be used for personalized medicine, namely adjusting and customizing healthcare with decisions and practices being suitable to the individual patient by use of any additional information collected at different stages of the disease.
  • the invention further provides a simple approach for assessing responsiveness of a mammalian subject to a specific treatment with a therapeutic agent as indicated above, or evaluating the efficacy of the specific treatment on a subject suffering from a chronic inflammatory condition.
  • the method of the invention may comprise:
  • a determining the level of expression of CD247 in a biological sample of the subject to obtain a CD247 expression value in the tested biological sample. It should be noted that the sample is obtained prior to initiation of said treatment.
  • step (b) determining the level of expression of CD247 in at least one other biological sample of the subject to obtain a CD247 expression value in the sample. It should be noted that the at least one other sample is obtained after or during the initiation of the specific treatment.
  • Step (c) involves comparing CD247 expression value in the biological sample obtained in step (a), with at least one CD247 expression value obtained in step (b).
  • the level of CD247 expression in a sample should be also compared to a predetermined standard levels of expression calculated for a population of age and gender matched healthy subjects. This comparison should be performed against a CD247 cut off value or standard curve of control/healthy individual expression levels or responders exhibiting the same pathology.
  • a higher CD247 expression value in a sample obtained after initiation of the treatment according to step (b) as compared to the CD247 expression value in a sample obtained prior to initiation of said treatment according to step (a), and compared with the standard curves of control/healthy individual expression levels or responders exhibiting the same pathology, is indicative of a successful therapy.
  • the method of the invention is based on determining the expression level of a specific biomarker, CD247, in a sample.
  • level of expression or “expression level” are used interchangeably and generally refer to the amount of a polynucleotide or a protein in a biological sample.
  • Expression generally refers to the process by which gene-encoded information is converted into the structures present and operating in the cell. Therefore, according to the invention "expression" of a gene, specifically, a gene encoding CD247 may refer to transcription into a polynucleotide, translation into a protein, or even posttranslational modification of the protein.
  • Fragments of the transcribed polynucleotide, the translated protein, or the post-translationally modified protein shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-trans lational processing of the protein, e.g., by proteolysis.
  • expression level is reflected by measurement and determination of an expression value.
  • expression value level of expression or expression level refers to numerical representation of a quantity of a gene product, which herein is a protein, but may also be an mRNA.
  • a subject will be considered suitable for treatment when the expression value of CD247 (or as will be explained herein after, a normalized expression value of CD247) determined in a biological sample obtained from said subject is equal, within the range of or above ("positive") a corresponding predetermined (normalized) expression value of CD247 obtained for a population of patients exhibiting a beneficial effect in response to treatment with the same therapeutic agent (a successful treatment).
  • a corresponding predetermined (normalized) expression value of CD247 obtained for a population of patients exhibiting a beneficial effect in response to treatment with the same therapeutic agent (a successful treatment).
  • control samples are used instead of- or in addition to- a predetermined standard (cutoff)
  • the expression value of the sample is compared to the expression values obtained for the control sample.
  • a higher expression value of CD247 in response to a certain treatment is determined as "positive" rate of change and a lower expression value of CD247 in response to said treatment (down-regulated expression) is determined as "negative" rate of change.
  • up-regulated expression a higher expression value of CD247 in response to a certain treatment
  • down-regulated expression a lower expression value of CD247 in response to said treatment
  • a "positive”, “higher” or “up-regulated” expression value of CD247, or rate of change in the expression of CD247 in response to treatment determined in said biological sample indicates that the subject is suitable for the specific treatment and exhibits a beneficial response.
  • the specific therapeutic agent is suitable and effective to the particular examined patient, where the expression value (specifically, the normalized expression values or rate of change) of CD247 determined in the biological sample is in the range of the normalized expression value of CD247 obtained in a suitable control sample or a corresponding predetermined standard expression value (cutoff values) of CD247, specifically, a standard rate of responder population, by at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11 %, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21 %, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31 %, at least
  • Example 2 ( Figure 9). This example demonstrates the rate of change in an expression value of CD247 determined in a biological sample during a treatment with doxorubicin, where an increase of about 24% in the expression value of CD247 was obtained upon treatment with doxorubicin, indicative of the suitability of said treatment to said subject.
  • Example 2 ( Figure 11) demonstrates an expression value of CD247 determined in a biological sample during a treatment with busulfan, where an increase of about 23% in the expression value of CD247 was obtained upon treatment with this agent, indicative of the suitability of said treatment to said patient.
  • CD247 expression values (specifically, normalized expression values or rate of change in the expression of CD247) that are determined as "negative” as being lower than the normalized expression value of CD247 obtained in a suitable control sample, or a corresponding predetermined standard expression value (cut-off) of CD247, correlate with an aggravation of the chronic inflammation condition or immune-suppressive environment of said patient in response to treatment with a specific therapeutic agent, and therefore indicate that this specific examined therapeutic agent may not be suitable for treating the specific subject that suffers from a specific chronic inflammatory condition.
  • Such control or cutoff value may be obtained from a population of age and gender matched healthy subjects and/or a population of subjects suffering from the same pathologic condition that respond well to the same treatment (responders).
  • such "negative" expression value or rate of change in response to a specific treatment should be in the range (+/- SD) of cutoff value determined for a population of subjects suffering from the same pathological disorder that are considered as not responding to the treatment, indicating that the examined patient belongs to said population.
  • CD247 in response to treatment with a therapeutic agent is correlated with an improvement in the chronic inflammation status, and thereby indicates that the tested subject would respond and exhibit a beneficial response to treatment with the specific therapeutic agent.
  • “high level of expression”, or “positive” expression values, or rate of change, when applicable, as used herein for CD247 denotes a level significantly (e.g. as determined by statistical determination) higher than the expression before treatment that is in the range of a predetermined standard. It should be noted that such standard value is predetermined for a population of patients suffering from the same pathologic condition that perform beneficial effect "responders". Therefore, expression value or rate of change that is within the range of such cutoff indicates that the examined subject belongs to a population of responders.
  • Standard or a “predetermined standard” as used herein, denotes either a single standard value or a plurality of standards with which the level of CD247 expression from the tested sample is compared.
  • the standards may be provided, for example, in the form of discrete numeric values or is calorimetric in the form of a chart with different colors or shadings for different levels of expression; or they may be provided in the form of a comparative curve prepared on the basis of such standards (standard curve).
  • the standards may be prepared by determining the level of expression of CD247 present in a sample obtained from a plurality of patients that were diagnosed or determined (by other means, for example by a physician, by histological techniques etc.) as performing a beneficial response ("responders") to a certain treatment and a population of patients that do not respond well to the same therapeutic agent (non- responders, being correlated with a low level of expression of CD247).
  • the level of expression for the preparation of the standards may also be determined by various conventional methods known in the art. The methods of the invention may be carried out in parallel to a number of standards of healthy subjects and subjects of different chronic inflammatory condition states that respond or not respond to a certain treatment and the level determined in the assayed sample is then compared to such standards.
  • step (d) and in some embodiments, step (b), of the method for determining the efficacy of a treatment with a therapeutic agent on a subject suffering from a chronic inflammatory condition provided by the invention refers to a predetermined standard expression value (step b), or when applicable (in step d), a predetermined standard rate of change, that in other words may be defined as predetermined "cutoff values.
  • a "cutoff value” sometimes referred to simply as “cutoff” herein, is a value that meets the requirements for both high sensitivity (true negative rate) and high specificity (true positive rate).
  • CD247 expression level values that are higher ("positive") or lower ("negative") in comparison with said CD247 expression corresponding cutoff value or a predetermined standard expression value or rate of change, indicate that the examined sample belongs to a pre- established population associated with a specific chronic inflammation rate in response to a specific treatment with a therapeutic agent (low or high, respectively) and limited to the said sensitivity and specificity.
  • the specific cutoff value reflects the threshold, indicating whether a specific population exhibits a beneficial response to a certain treatment, or in contrast, exhibit a harmful effect in response to the same treatment.
  • sensitivity and “specificity” are used herein with respect to the ability of the biomarker of the invention, CD247, to correctly classify a sample as belonging to a pre-established population associated with a specific probability to respond (exhibit a beneficial effect) to a specific therapeutic agent, and thereby to determine whether a certain therapeutic agent is suitable for treatment as leading to a beneficial effect, or alternatively, is not suitable for treatment of the particular pathologic condition, as leading to a deleterious effect on the immune system of the treated subject.
  • “Sensitivity” and “specificity” indicate the performance of the biomarker of the invention, CD247, with respect to correctly classifying samples as belonging to pre- established populations that are likely to display a beneficial effect in response to a certain treatment ("responders”), or alternatively, to correctly classify the sample a belonging to pre-established populations of "non-responders” to the specific agent that display a chronic inflammation status.
  • CD247 expression values are within the range of a cutoff value defined for a population of "responders”, that is, "positive” values indicating elevated expression of CD247 in response to the particular treatment reflecting improvement in the chronic inflammation states, more specifically, indicating that the diseased subject is more likely to display a beneficial response to the specific therapeutic agent, that leads to reduction, and preferably, elimination of the chronic inflammation than corresponding pre-established populations wherein said corresponding CD247 expression values in response to treatment with the same therapeutic agent, are lower than the cutoff predetermined for responsive population or healthy controls (age and gender matched), that is, "negative” values indicating lower beneficial effect, and thus, higher chronic inflammation rates.
  • sensitivity relates to the rate of correct identification of high-chronic inflammation rate samples that reflect a non suitable therapeutic agent, as such out of a group of samples (true negatives)
  • specificity relates to the rate of correct identification of low-inflammation rate samples reflecting a suitable treatment as such out of a group of samples, in a reproducible manner (true positives).
  • Cutoff values may be used as a control sample, said cutoff values being the result of a statistical analysis of CD247 expression value differences in pre-established populations with either a chronic inflammation state, reflecting "non-responsiveness” or deleterious effects of the specific therapeutic agent or alternatively, populations of "responsive” or “healthy” subjects with no inflammation or reduced inflammation in response to a successful treatment.
  • CD247 expression values or rate of change in response to treatment with a certain therapeutic agent that are lower than the cutoff value found by the inventors (i.e., "negative” expression value)
  • a certain therapeutic agent that are lower than the cutoff value found by the inventors
  • the expression value falls within the range of the cutoff value (i.e., "positive” results).
  • a given population treated with a specific therapeutic agent having specific clinical parameters will have a defined likelihood to respond or alternatively, not respond (developing chronic inflammation) based on the expression values of CD247 that may be within the range of or below said cutoff values.
  • the nature of the invention is such that the accumulation of further patient and/or healthy donors data may improve the accuracy of any obtained cutoff values, which are usually based on an ROC (Receiver Operating Characteristic) curve generated according to accumulated patient and/or healthy donors data using, for example, a commercially available analytical software program.
  • ROC Receiveiver Operating Characteristic
  • the CD247 expression values are selected along the ROC curve for optimal combination of sensitivity and specificity, which are as close to 100% as possible, and the resulting values are used as the cutoff values that distinguish between patients who will display chronic inflammation at a certain rate in response to a specific treatment, and those who will display a beneficial response to the same therapeutic agent that is reflected by reduced inflammation (with said given sensitivity and specificity).
  • the ROC curve may evolve as more and more patient-chronic inflammation and healthy donor data and related CD247 expression values are recorded and taken into consideration, modifying the optimal cutoff values and improving sensitivity and specificity.
  • any cutoff values should be viewed as a starting point that may shift as more patient-chronic inflammation data in response to a specific treatment allows more accurate cutoff value calculation.
  • control samples or standard curves are age and gender matched subjects. It should be further appreciated that standard curves for "responders" may be prepared specifically for each pathologic condition, and in some embodiments, also for each therapeutic agent (or treatment regimen).
  • the inventors have analyzed the expression values of CD247 in response to treatment with a certain therapeutic agent, further, in order to discover specific cutoff values, a deviation from which is indicative of an increased likelihood for reduced chronic inflammation and immuno-suppression state in response to treatment with a specific therapeutic agent of a tested subject that suffers from a chronic inflammatory condition, thereby determining the efficacy of a specific treatment with a certain therapeutic agent.
  • an important step in the method of the invention is determining whether the expression value of CD247 (specifically, normalized) is "positive” and thereby belongs to a pre-established population with an associated specific likelihood of exhibiting a beneficial effect in response to the specific agent, or is "negative” and thereby belongs to a pre-established population with an elevated or unimproved chronic inflammation state (non- responsive).
  • Such step involves calculating and measuring the difference between (thereby comparing) the expression values of the examined sample and the cutoff value and determining whether the examined sample can be defined as positive or negative.
  • CD247 with a "positive" normalized expression value in response to treatment with a specific therapeutic agent indicates that the subject belongs to a pre-established population with an associated reduced chronic inflammation state which is lower than the chronic inflammation state associated with, ceteris paribus, subjects where CD247 have “negative” normalized expression values (or rate of change, when applicable) in response to the same treatment, indicates a reduced expression of CD247 and an elevated chronic inflammation state.
  • a specific therapeutic agent or rate of change, when applicable
  • a "positive result" may be determined where a normalized value of CD247 is elevated in response to treatment and therefore, the expression value of the sample is within the range [+/- SD (Standard Deviation)] of the cutoff value and therefore predicts reduction and sometimes elimination of the chronic inflammation and associated immunosuppression, in response to treatment with a specific agent, indicating the suitability of the treatment for the specific subject.
  • a decrease below a predetermined cutoff or threshold reflects a decrease or no improvement in CD247 expression in response to a certain treatment (that is a reduced or non existing rate of change), determined in the sample of the subject suffering from a chronic inflammatory condition relative to the reference expression or rate of change in the expression in response to treatment, which is lower than a predetermined cutoff or threshold such as about 10 %, e.g., lower than about 20 %, e.g., lower than about 30 %, e.g., lower than about 40 %, e.g., lower than about 50 %, e.g., lower than about 60 %, lower than about 70 %, lower than about 80 %, lower than about 90 %, lower than about 2 times, lower than about three times, lower than about four time, lower than about five times, lower than about six times, lower than about seven times, lower than about eight times, lower than about nine times, lower than about 20 times, lower than about 50 times, lower than about
  • an identical rate of change (within the range of) in the expression of CD247 in response to such treatment, or a change above a predetermined cutoff or threshold in the ratio determined in the sample of the treated subject that suffers from a chronic inflammatory condition as compared to the reference rate of change indicates the reduction or elimination of the chronic inflammation, and thereby, a successful treatment with the specific therapeutic agent.
  • More specific embodiments for such elevation may include about 10 %, e.g., higher than about 20 %, e.g., higher than about 30 %, e.g., higher than about 40 %, e.g., higher than about 50 %, e.g., higher than about 60 %, higher than about 70 %, higher than about 80 %, higher than about 90 %, higher than about 2 times, higher than about three times, higher than about four time, higher than about five times, higher than about six times, higher than about seven times, higher than about eight times, higher than about nine times, higher than about 20 times, higher than about 50 times, higher than about 100 times, higher than about 200 times, higher than about 350, higher than about 500 times, higher than about 1000 times, or more relative to the reference rate of change.
  • the reduction in the expression of CD247 in response to a treatment with a specific therapeutic agent constitutes an early marker of a deleterious effect of the treatment (or non-responsiveness to a given treatment) reflected by an elevated inflammatory state that may indicate the regression or recurrence of a chronic inflammatory condition.
  • medical staff may become aware of a regression or recurrence of the diseases earlier than they currently are, and consequently, provide earlier and more effective treatment.
  • decrease or reduction may be a decrease or reduction of between about 10% to 100% of the expression of such biomarker.
  • decrease or reduction may be a decrease or reduction of between about 10% to 100% of the expression of such biomarker.
  • the terms “decrease”, “reduction” and “elimination” as used herein relate to the act of becoming progressively smaller in size, amount, number, or intensity. Particularly, a reduction of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the expression as compared to a suitable control. It should be further noted that decrease or reduction may be also a decrease of about 2 to 100 folds.
  • the decrease of the levels or expression of said CD247 might be either in the transcription, translation or the stability of said biomarker.
  • percentage values such as, for example, 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change" values, i.e., 0.1 , 0.5, 1.2, 5, etc., respectively.
  • the invention relates to a method for monitoring and assessing the efficacy of a treatment with a therapeutic agent and thereby providing information concerning the disease progression.
  • the therapeutic agent may lead to either a direct or indirect anti-/pro-inflammatory effect, and therefore, CD247 levels reflect the effect of the therapeutic agent on the immune-system of the diseased subject.
  • such method comprises the steps of: In a first step (a) determining the level of expression CD247 in a biological sample of the examined subject to obtain an expression value. It should be appreciated that in certain embodiments, this sample may be obtained either prior to or at any time after the initiation of the treatment with the examined therapeutic agent.
  • Step (b) involves repeating step (a) to obtain expression values of CD247, for at least one more temporally separated test sample. It should be noted that at least one of the temporally separated samples must be obtained after the initiation of the treatment.
  • step (c) calculating the rate of change of the expression values of CD247 between the temporally separated test samples.
  • step (d) determining if the rate of change obtained in step (c) is any one of, positive, negative or equal to a predetermined standard rate of change determined between at least two temporally separated samples.
  • the rate of change obtained in step (c) is compared with a predetermined standard rate of change (cutoff or standard curves) determined between at least one sample obtained prior to and at least one sample obtained following interferon treatment.
  • this step involves determining if the rate of change obtained in step (c) is any one of, positive, negative or equal to the rate of change calculated for expression values in the control samples obtained from at least two temporally separated samples of subjects suffering from the same pathologic condition that are considered as "responders" and from subjects considered as "non-responders", wherein at least one sample of said at least two samples is obtained after the initiation of the specific treatment.
  • a "positive" rate of change of CD247 expression value indicates that the tested subject displays rate of change values that are within the range of a predetermined cutoff for subjects suffering from the same condition that are classified as “responders” or in the range of expression of healthy donors.
  • rate of change values that are within the range of a predetermined cutoff for subjects suffering from the same condition that are classified as “responders” or in the range of expression of healthy donors.
  • response refers to an improvement in at least one relevant clinical parameter as compared to an untreated subject diagnosed with the same pathology (e.g., the same type, stage, degree and/or classification of the chronic inflammatory condition), or as compared to the clinical parameters of the same subject prior to said treatment.
  • non responder or “non-responsive” to treatment using a specific therapeutic agent, refers to a patient not experiencing an improvement in at least one of the clinical parameter and is diagnosed with the same condition as an untreated subject diagnosed with the same pathology (e.g., the same type, stage, degree and/or classification of the chronic inflammatory condition), or experiencing the clinical parameters of the same subject prior to such treatment.
  • pathology e.g., the same type, stage, degree and/or classification of the chronic inflammatory condition
  • samples taken in more time -points may provide a statistically robust analysis of said expression trends, and may also be utilized as a method for continuous monitoring of subjects, especially those still undergoing and those that have undergone therapy.
  • the number of samples collected and used for evaluation of the subject may change according to the frequency with which they are collected.
  • the samples may be collected at least every day, every two days, every four days, every week, every two weeks, every three weeks, every month, every two months, every three months every four months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every year or even more.
  • the rate of change may be calculated as an average rate of change over at least three samples taken in different time points, or the rate may be calculated for every two samples collected at adjacent time points.
  • the sample may be obtained from the monitored patient in the indicated time intervals for a period of several months or several years. More specifically, for a period of 1 year, for a period of 2 years, for a period of 3 years, for a period of 4 years, for a period of 5 years, for a period of 6 years, for a period of 7 years, for a period of 8 years, for a period of 9 years, for a period of 10 years, for a period of 11 years, for a period of 12 years, for a period of 13 years, for a period of 14 years, for a period of 15 years or more.
  • At least two "temporally- separated" test samples must be collected from the treated patient, and preferably more.
  • the expression of at least CD247 is then determined using the method of the invention, applied for each sample.
  • the rate of change in this biomarker expression is then calculated by determining the difference in expression values (specifically, normalized values) of said CD247 between any two samples obtained from the same patient in different time -points or time intervals.
  • time interval This period of time, also referred to as “time interval”, or the difference between time points (wherein each time point is the time when a specific sample was collected) may be any period deemed appropriate by medical staff and modified as needed according to the specific requirements of the patient and the clinical state he or she may be in. It should be noted that such interval could be as indicated herein above.
  • a calculated or average negative rate of change of the normalized expression values of CD247 indicates that the subject is in a chronic inflammation state in response to said treatment.
  • the diagnostic and prognostic method of the invention in order to execute the diagnostic and prognostic method of the invention, at least two different samples, and preferably, more than two, must be obtained, from the subject in order to calculate the rate of change in the expression of CD247 in response to treatment with a specific compound.
  • the prognostic method of the invention may be applicable for early, sub-symptomatic diagnosis of chronic inflammation in a treated subject, when used for analysis of more than a single sample along the time-course of diagnosis, treatment and follow-up.
  • An “early diagnosis” provides diagnosis prior to appearance of clinical symptoms.
  • Prior as used herein is meant days, weeks, months or even years before the appearance of such symptoms. More specifically, at least 1 week, at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or even few years before clinical symptoms appear.
  • CD247 a decline in the expression of CD247 indicates a chronic inflammation associated with immunosuppression that is a result of non-responsiveness reflecting failure of a treatment and may provide an early sign before over symptoms occur, allowing for a quicker and more efficient therapeutic response.
  • the invention therefore further provides a prognostic method.
  • Prognosis is defined as a forecast of the future course of a disease or disorder, based on medical knowledge. This highlights the major advantage of the instant invention over prior art, namely, the ability to predict chronic inflammation indicating regression or recurrence of the diseases in patients treated with a non appropriate therapeutic agent. This early prognosis facilitates the selection of appropriate treatment regimens that may minimize the regression or recurrence of the diseases, individually to each patient, as part of personalized medicine.
  • the invention thus provides a method for predicting the efficacy and suitability of a specific treatment.
  • the phrase "predicting or evaluating efficacy of a treatment” refers to determining the likelihood that a specific treatment using a therapeutic agent is efficient or non-efficient in treating the chronic inflammatory condition, e.g., the success or failure of the treatment in treating the chronic inflammatory condition in a subject in need thereof. More specifically, a treatment with a therapeutic agent that directly, as an anti-inflammatory agent, or indirectly (a chemotherapeutic or biological agent) affects inflammation.
  • the term "efficacy” as used herein refers to the extent to which the antiinflammatory treatment produces a beneficial result, e.g., an improvement in one or more symptoms of the pathology (caused by the chronic inflammatory condition) and/or clinical parameters related to the pathology as described herein below.
  • the efficacy of an anti-inflammatory treatment may be evaluated using standard therapeutic indices for chronic inflammatory condition, for example, a proliferative disorder, an autoimmune disease or an infectious disease).
  • the efficacy of treatment is a long- term efficacy.
  • long-term efficacy refers to the ability of a treatment to maintain a beneficial result over a period of time, e.g., at least about 16 weeks, at least about 26 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 48 weeks, at least about 52 weeks, at least about 18 months, at least about 24 months, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, or longer.
  • a treatment with a therapeutic agent that either directly or indirectly affects inflammation is considered efficient in treating a chronic inflammatory condition if it exerts an improvement in at least one relevant clinical parameter related to said condition in the treated subject as compared to an untreated subject diagnosed with the same condition (e.g., where the chronic inflammatory condition is cancer, such parameter include the type, stage, degree and/or classification of the solid tumor), or as compared to the clinical parameters related to the said condition of the same subject prior to the anti- inflammatory treatment.
  • a "positive clinical outcome” and “beneficial response” can be assessed using any endpoint indicating a benefit to the patient, including, without limitation, (1) inhibition, to some extent, of tumor growth, including slowing down and complete growth arrest; (2) reduction in the number of tumor cells; (3) reduction in tumor size; (4) inhibition (i.e., reduction, slowing down or complete stopping) of tumor cell infiltration into adjacent peripheral organs and/or tissues; (5) inhibition of metastasis; (6) enhancement of anti-tumor immune response, possibly resulting in regression or rejection of the tumor; (7) relief, to some extent, of one or more symptoms associated with the tumor; (8) increase in the length of survival following treatment; and/or (9) decreased mortality at a given point of time following treatment.
  • Positive clinical response may also be expressed in terms of various measures of clinical outcome. Positive clinical outcome can also be considered in the context of an individual's outcome relative to an outcome of a population of patients having a comparable clinical diagnosis, and can be assessed using various endpoints such as an increase in the duration of Recurrence-Free interval (RFI), an increase in the time of survival as compared to Overall Survival (OS) in a population, an increase in the time of Disease-Free Survival (DFS), an increase in the duration of Distant Recurrence- Free Interval (DRFI), and the like.
  • RFI Recurrence-Free interval
  • OS Overall Survival
  • DFS Disease-Free Survival
  • DRFI Distant Recurrence- Free Interval
  • An increase in the likelihood of positive clinical response corresponds to a decrease in the likelihood of cancer recurrence.
  • the invention provides a prognostic tool for detecting responders vs. non- responders to a given immune-based therapy.
  • the method of the invention is applicable for determining the suitability and efficacy of a specific therapeutic agent or combinations of agents.
  • the method of the invention may further provide information regarding the overall treatment regimen. More specifically, by sensing changes in the inflammatory state as reflected by changes in the expression levels of CD247, the invention may enable defining (or fine tuning) the optimal concentrations of the therapeutic agent and providing information regarding the preferable duration of treatment. As demonstrated by Figure 20, the use high concentration of the 5FU+CPT11, resulted in a harmful effect, however, when low doses of the same combination were applied, this combination displayed a beneficial effect. As indicated above, the method of the invention is based on determining the expression of the biomarker CD247 in a sample.
  • the determination of the level of expression of CD247 in a biological sample of the tested subject may be performed by a method comprising the step of contacting detecting molecules specific for CD247 with a biological sample of said subject, or with any nucleic acid or protein product obtained there from. More specifically, the method of invention relies on the detection of CD247 by contacting detecting molecules specific for CD247 with a biological sample.
  • the term "contacting" means to bring, put, incubate or mix together. As such, a first item is contacted with a second item when the two items are brought or put together, e.g., by touching them to each other or combining them.
  • the term "contacting" includes all measures or steps, which allow interaction between at least one detecting molecule specific for CD247 and the tested sample, or any component derived thereforom.
  • the method of the invention further involves contacting steps, which allow interaction between at least one detecting molecule specific for a suitable reference control and the tested sample or any component derived thereforom.
  • the contacting is performed in a manner by which the at least one of detecting molecule of CD247 and at least one suitable reference control can interact with or bind to the peptide molecules or nucleic acid molecule in the tested sample.
  • the binding will preferably be non-covalent, reversible binding, e.g., binding via salt bridges, hydrogen bonds, hydrophobic interactions or a combination thereof.
  • the method of the invention may further comprise an additional and optional step of normalization.
  • the expression levels of a reference control are also determined, and subsequently, the expression levels of CD247 may be normalized according to the expression value of said at least one reference control, in the same test sample.
  • similar normalization is performed also relative to a control sample or a representing standard when applicable.
  • a normalized expression value of CD247 in the test sample, and optionally relative to a control sample is obtained.
  • the next step in this embodiment involves comparing the normalized CD247 expression value in the test biological sample obtained in this additional step, with a predetermined standard expression value, or a cutoff value, or with a normalized expression value of CD247 in a control sample optionally obtained in this additional step.
  • a "negative" that is a lower expression value (or rate of change) of CD247 in the tested sample, as compared to a predetermined standard expression value (cutoff) or to the expression value of CD247 in a control sample, in response to treatment with a therapeutic agent is indicative of an unsuccessful treatment that leads to, increase or does not improve a chronic inflammation and associated immune- suppression in the tested subject.
  • the optional normalization step of the method of the invention involves normalization of the measured expression values of CD247, to obtain normalized expression value.
  • the measured levels of expression of CD247 are routinely normalized using data of expression levels of the control reference proteins.
  • normalization is a process by which a measurement raw data is converted into data that may be directly compared with other so normalized data.
  • measurements of marker genes or proteins, specifically, CD247, expression levels are prone to errors caused by, for example, unequal degradation of measured samples, different loaded quantities per assay, and other various errors. More specifically, any assayed sample may contain more or less biological material than is intended, due to human error and equipment failures.
  • control references used by the invention i.e., CD3s (for T cells) is equal and stable in samples displaying chronic inflammation in response to a certain therapeutic agent and in control samples of healthy donors or responsive subjects that do not display chronic inflammation and therefore exhibit a beneficial effect of the treatment.
  • other control references may be used, for example, any one of CD56 or SNX27.
  • method of the invention may further comprise the use of reference control.
  • the method further involves contacting the biological sample or any protein or nucleic acid product obtained therefrom with detecting molecules specific for at least one reference control.
  • the reference control protein used by the methods of the invention displays constant expression pattern in a sample of an un-treated subject that suffers from a chronic inflammatory condition, a treated subject exhibiting a beneficial response (also referred to herein as a "responder"), a subject that exhibits a deleterious response to the same therapeutic agent, as well as healthy subjects.
  • a beneficial response also referred to herein as a "responder”
  • CD3s is used as a control reference (specifically, for T cells).
  • such reference control may be at least one of CD3s, CD38, CD3y, TCRa, TCRP, CD56 and in some embodiments also TCR ⁇ and TCR ⁇ .
  • determination of the level of CD247 expression in the biological sample can be effected at the transcriptional level (i.e., mRNA) using detecting molecules that are based on nucleic acids (an oligonucleotide probe or primer), or alternatively, at the translational level (i.e. protein) using amino acid based detecting molecules, as also demonstrated by the present invention.
  • the detecting molecules used by the method of the invention may be isolated detecting amino acid molecules or isolated detecting nucleic acid molecules, or any combinations thereof.
  • the detection of CD247 expression can be affected at the protein level. Therefore, the detecting molecules used by the method of the invention may be amino acid molecules, specifically, an isolated antibodies that specifically recognize and binds CD247. As shown by the following Examples, the anti-CD247 antibodies used by the method of the invention are antibodies specifically directed to the C terminal domain of the CD247 molecule. However, it should be noted that according to certain embodiments, antibodies directed to any part or fragment of CD247 molecule may be used for performing the invention.
  • the detecting molecules of the invention may be amino acid based molecules that may be referred to as protein/s or polypeptide/s.
  • protein and “polypeptide” are used interchangeably to refer to a chain of amino acids linked together by peptide bonds.
  • a protein is composed of less than 200, less than 175, less than 150, less than 125, less than 100, less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, less than 10, or less than 5 amino acids linked together by peptide bonds.
  • a protein is composed of at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500 or more amino acids linked together by peptide bonds.
  • peptide bond as described herein is a covalent amid bond formed between two amino acid residues.
  • the invention further contemplates the use of amino acid based molecules such as proteins or polypeptides as detecting molecules disclosed herein and would be known by a person skilled in the art to measure the protein marker of the invention, CD247.
  • the detecting amino acid molecules are isolated antibodies, with specific binding selectively to CD247.
  • the level of expression of CD247 may be determined using an immunoassay which is selected from the group consisting of FACS, a Western blot, an ELISA, a RIA, a slot blot, a dot blot, immunohistochemical assay, immuno fluorescent assay and a radio-imaging assay.
  • antibody as used in this invention includes whole antibody molecules as well as functional fragments thereof, such as Fab, F(ab')2, and Fv that are capable of binding with antigenic portions of the target polypeptide, i.e. CD247.
  • the antibody is preferably monospecific, e.g., a monoclonal antibody, or antigen-binding fragment thereof.
  • monospecific antibody refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope. This term includes a "monoclonal antibody” or “monoclonal antibody composition”, which as used herein refer to a preparation of antibodies or fragments thereof of single molecular composition.
  • the antibody can be a human antibody, a chimeric antibody, a recombinant antibody, a humanized antibody, a monoclonal antibody, or a polyclonal antibody.
  • the antibody can be an intact immuno globulin, e.g., an IgA, IgG, IgE, IgD, lgM or subtypes thereof.
  • the antibody can be conjugated to a functional moiety (e.g., a compound which has a biological or chemical function.
  • the antibody used by the invention interacts with a polypeptide that is CD247, with high affinity and specificity.
  • antibody also encompasses antigen-binding fragments of an antibody.
  • antigen-binding fragment of an antibody (or simply “antibody portion,” or “fragment”), as used herein, may be defined as follows:
  • Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
  • Fab' the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;
  • Fv defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
  • Single chain antibody (“SCA”, or ScFv), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
  • Purification of serum immunoglobulin antibodies can be accomplished by a variety of methods known to those of skill in the art including, precipitation by ammonium sulfate or sodium sulfate followed by dialysis against saline, ion exchange chromatography, affinity or immuno-affinity chromatography as well as gel filtration, zone electrophoresis, etc.
  • the anti- CD247 antibodies used by the present invention may optionally be covalently or non- covalently linked to a detectable label.
  • labeled can refer to direct labeling of the antibody via, e.g., coupling (i.e., physically linking) a detectable substance to the antibody, and can also refer to indirect labeling of the antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody. More specifically, detectable labels suitable for such use include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include magnetic beads (e.g.
  • DYNABEADS fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, and the like), radio labels (e.g., 3 H, 125 I, 35 S, 14 C, or 32 P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA and competitive ELISA and other similar methods known in the art) and colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.
  • fluorescent dyes e.g., fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, and the like
  • radio labels e.g., 3 H, 125 I, 35 S, 14 C, or 32 P
  • enzymes e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in
  • radiolabels may be detected using photographic film or scintillation counters
  • fluorescent markers may be detected using a photodetector to detect emitted illumination
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
  • binding specificity specifically binds to an antigen
  • specifically immuno-reactive with specifically directed against or “specifically recognizes”
  • binding reaction which is determinative of the presence of the epitope in a heterogeneous population of proteins and other biologies.
  • “selectively bind” in the context of proteins encompassed by the invention refers to the specific interaction of a any two of a peptide, a protein, a polypeptide an antibody, wherein the interaction preferentially occurs as between any two of a peptide, protein, polypeptide and antibody preferentially as compared with any other peptide, protein, polypeptide and antibody.
  • epitope is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody.
  • Epitopes or “antigenic determinants” usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.
  • the specified antibodies bind to a particular epitope at least two times the background and more typically more than 10 to 100 times background.
  • selective binding means that a molecule binds its specific binding partner with at least 2-fold greater affinity, and preferably at least 10-fold, 20-fold, 50-fold, 100-fold or higher affinity than it binds a non-specific molecule.
  • immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein or carbohydrate.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein or carbohydrate.
  • the expression level of the CD247 protein, in the tested sample can be determined using different methods known in the art, specifically method disclosed herein below as non- limiting examples.
  • This method involves fixation of a sample containing a protein substrate (e.g., fixed cells or a proteinaceous solution) to a surface such as a well of a microtiter plate.
  • a protein substrate e.g., fixed cells or a proteinaceous solution
  • a substrate-specific antibody coupled to an enzyme is applied and allowed to bind to the substrate. Presence of the antibody is then detected and quantitated by a colorimetric reaction employing the enzyme coupled to the antibody.
  • Enzymes commonly employed in this method include horseradish peroxidase and alkaline phosphatase. If well calibrated and within the linear range of response, the amount of substrate present in the sample is proportional to the amount of color produced.
  • a substrate standard is generally employed to improve quantitative accuracy.
  • This method involves separation of a substrate from other protein by means of an acrylamide gel followed by transfer of the substrate to a membrane (e.g., nitrocellulose, nylon, or PVDF). Presence of the substrate is then detected by antibodies specific to the substrate, which are in turn detected by antibody-binding reagents.
  • Antibody-binding reagents may be, for example, protein A or secondary antibodies. Antibody-binding reagents may be radiolabeled or enzyme-linked, as described hereinafter. Detection may be by autoradiography, colorimetric reaction, or chemiluminescence. This method allows both quantitation of an amount of substrate and determination of its identity by a relative position on the membrane indicative of the protein's migration distance in the acrylamide gel during electrophoresis, resulting from the size and other characteristics of the protein.
  • this method involves precipitation of the desired protein (i.e., the substrate) with a specific antibody and radiolabeled antibody-binding protein (e.g., protein A labeled with I ) immobilized on a precipitable carrier such as agarose beads.
  • a specific antibody and radiolabeled antibody-binding protein e.g., protein A labeled with I
  • the radio-signal detected in the precipitated pellet is proportional to the amount of substrate bound.
  • a labeled substrate and an unlabelled antibody-binding protein are employed.
  • a sample containing an unknown amount of substrate is added in varying amounts.
  • the number of radio counts from the labeled substrate-bound precipitated pellet is proportional to the amount of substrate in the added sample.
  • This method involves detection of a substrate in situ in cells bound by substrate- specific, fluorescently labeled antibodies.
  • the substrate- specific antibodies are linked to fluorophores. Detection is by means of a flow cytometry machine, which reads the wavelength of light emitted from each cell as it passes through a light beam.
  • This method may employ two or more antibodies simultaneously, and is a reliable and reproducible procedure used by the present invention.
  • the substrate specific antibodies may be enzyme-linked or linked to fluorophores. Detection is by microscopy, and is either subjective or by automatic evaluation. With enzyme-linked antibodies, a calorimetric reaction may be required. It will be appreciated that immunohistochemistry is often followed by counterstaining of the cell nuclei, using, for example, Hematoxyline or Giemsa stain.
  • the detecting molecules for CD247 expression may be isolated detecting nucleic acid molecules.
  • such detecting nucleic acid molecules may be isolated oligonucleotides, each oligonucleotide specifically hybridizes to a nucleic acid sequence of the RNA products of said CD247.
  • the oligonucleotide used as a detecting molecule according to certain embodiments of the invention may be any one of a pair of primers or nucleotide probe.
  • the level of expression of CD247 may be determined using a nucleic acid amplification assay selected from the group consisting of: a Real-Time PCR, micro arrays, PCR, in situ Hybridization and Comparative Genomic Hybridization. It should be noted that in particular embodiments, the invention further encompasses the use of aptamers as a nucleic acid based detection molecules that specifically recognize and bind the CD247 protein.
  • nucleic acids is interchangeable with the term “polynucleotide(s)” and it generally refers to any polyribonucleotide or poly-deoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA or any combination thereof.
  • Nucleic acids include, without limitation, single- and double- stranded nucleic acids.
  • nucleic acid(s) also includes DNAs or RNAs as described above that contain one or more modified bases.
  • oligonucleotide is defined as a molecule comprised of two or more deoxyribonucleotides and/or ribonucleotides, and preferably more than three. Its exact size will depend upon many factors which in turn, depend upon the ultimate function and use of the oligonucleotide.
  • the oligonucleotides may be from about 8 to about 1 ,000 nucleotides long. More specifically, the detecting oligonucleotides molecule used by the composition of the invention may comprise any one of 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 bases in length.
  • the expression level of the CD247 RNA product, in the tested sample can be determined using methods known in the art. Different methods of determining levels of the expression of the biomarker of the invention, CD247 (i.e., RNA or protein) in biological samples using nucleic acid base detection methods may be applicable for performing the method of the invention. Such procedures include, but are not limited to, Northern Blot Analysis, Polymerase chain reaction (PCR)-based methods (e.g., RT-PCR, using oligonucleotide primers or probes as the detection molecules),
  • PCR Polymerase chain reaction
  • RNA In Situ Hybridization Stain for example, the Affymetrix® GeneChip® Micro array
  • Oligonucleotide Microarray procedures for example, the Affymetrix® GeneChip® Micro array
  • detecting molecules either nucleic acid based or amino acid based
  • compositions and kits compositions and kits will be described herein after
  • isolated molecules when used in reference to a nucleic acid means that a naturally occurring sequence has been removed from its normal cellular (e.g., chromosomal) environment or is synthesized in a non-natural environment (e.g., artificially synthesized).
  • an "isolated” or “purified” sequence may be in a cell-free solution or placed in a different cellular environment.
  • purified does not imply that the sequence is the only nucleotide present, but that it is essentially free (about 90-95% pure) of non- nucleotide material naturally associated with it, and thus is distinguished from isolated chromosomes.
  • isolated and purified in the context of a proteinaceous agent (e.g., a peptide, polypeptide, protein or antibody) refer to a proteinaceous agent which is substantially free of cellular material and in some embodiments, substantially free of heterologous proteinaceous agents (i.e. contaminating proteins) from the cell or tissue source from which it is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • substantially free of cellular material includes preparations of a proteinaceous agent in which the proteinaceous agent is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • a proteinaceous agent that is substantially free of cellular material includes preparations of a proteinaceous agent having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous proteinaceous agent (e.g. protein, polypeptide, peptide, or antibody; also referred to as a "contaminating protein").
  • heterologous proteinaceous agent e.g. protein, polypeptide, peptide, or antibody; also referred to as a "contaminating protein”
  • the proteinaceous agent is recombinantly produced, it is also preferably substantially free of culture medium, i.e. culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation.
  • the proteinaceous agent When the proteinaceous agent is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the proteinaceous agent. Accordingly, such preparations of a proteinaceous agent have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the proteinaceous agent of interest. Preferably, proteinaceous agents disclosed herein are isolated.
  • the correlation of increased expression of CD247 with a beneficial effect of the examined therapeutic agent was further examined and established by examining other parameters involved with chronic inflammation. More specifically, increased population of myeloid derived suppressor cells (MDSCs), increase in NO and ROS, increased levels of S100A8, S100A9 proteins and reduced levels of cleaved casepase 3 are correlated with changes in the inflammatory environment.
  • MDSCs myeloid derived suppressor cells
  • S100A8 S100A9 proteins
  • reduced levels of cleaved casepase 3 are correlated with changes in the inflammatory environment.
  • the expression of CD247 seemed to be a more accurate and sensitive marker, the combination of several markers may increase sensitivity and specificity.
  • the method of the invention may further combine other markers and therefore comprise the step of at least one of:
  • MDSCs myeloid-derived suppressor cells
  • NO nitric oxide
  • ROS reactive oxygen species
  • the method of the invention is specifically applicable for determining the efficacy or suitability of a specific therapeutic agent for treating a chronic inflammatory condition, or generally, an immune -related disorder.
  • an "Immune -related disorder” is a condition that is associated with the immune system of a subject, either through activation or inhibition of the immune system, or that can be treated, prevented or diagnosed by targeting a certain component of the immune response in a subject, such as the adaptive or innate immune response.
  • the method of the invention provides determining the efficacy of treatment with a specific therapeutic agent on a subject suffering from any pathologic condition that may directly or indirectly lead to a chronic inflammatory condition, specifically to a systemic chronic inflammatory condition (and not only to a local inflammation).
  • such disorder may be a proliferative disorder, a chronic inflammatory condition, specifically, any one of an inflammatory disease, viral infections, an autoimmune disease, metabolic disorders and cancer.
  • the method of the invention may be specifically suitable for determining the efficacy of a specific therapeutic agent for treating an inflammatory disease or an inflammatory-associated condition that may be caused by any pathological disorder.
  • inflammatory disease or "inflammatory- associated condition” refers to any disease or pathologically condition which can benefit from the reduction of at least one inflammatory parameter, for example, induction of an inflammatory cytokine such as IFN-gamma, TNFa, ILip, IL-6 and IL-2.
  • the condition may be caused (primarily) from inflammation, or inflammation may be one of the manifestations of the diseases caused by another physiological cause.
  • the present invention provides a powerful tool for determining the suitability for treatment of a patient suffering from a disease associated with a chronic inflammatory condition.
  • a chronic inflammatory condition may be any one of a proliferative disorder, an autoimmune disease and an infectious disease.
  • the method of the invention may be applicable for chronic inflammatory condition that may be a proliferative disorder, an autoimmune disorder, an infectious disease, metabolic disorders and cancer.
  • chronic inflammatory state is reflected by an inflammatory response.
  • inflammatory response refers to an immune response which results in either chronic or acute inflammation, typically occurring as a result of injurious stimuli including infection, burns, trauma, neoplasia, autoimmune signals and exposure to chemicals, heat or cold or any other harmful stimulus.
  • An inflammatory response according to the present invention refers to a chronic inflammation.
  • the method of the invention may be used for determining the suitability of a certain therapeutic agent, specifically, a chemotherapeutic agent, biological agent or immuno-therapeutic agent, for treatment of a patient suffering from a chronic inflammatory condition that may be a proliferative disorder.
  • a certain therapeutic agent specifically, a chemotherapeutic agent, biological agent or immuno-therapeutic agent
  • the method of the invention may be specifically efficient for a proliferative disorder.
  • proliferative disorder As used herein to describe the present invention, "proliferative disorder”, “cancer”, “tumor” and “malignancy” all relate equivalently to a hyperplasia of a tissue or organ. If the tissue is a part of the lymphatic or immune systems, malignant cells may include non-solid tumors of circulating cells. Malignancies of other tissues or organs may produce solid tumors. In general, the methods of the present invention may be applicable for determining the suitability for treatment of a patient suffering from any one of non-solid and solid tumors.
  • Malignancy as contemplated in the present invention may be any one of carcinomas, melanomas, lymphomas, leukemias, myeloma and sarcomas.
  • Carcinoma as used herein, and will be described in more detail herein after in connection with colorectal carcinoma (CRC), refers to an invasive malignant tumor consisting of transformed epithelial cells. Alternatively, it refers to a malignant tumor composed of transformed cells of unknown histogenesis, but which possess specific molecular or histological characteristics that are associated with epithelial cells, such as the production of cytokeratins or intercellular bridges.
  • CRC colorectal carcinoma
  • Melanoma as used herein, is a malignant tumor of melanocytes.
  • Melanocytes are cells that produce the dark pigment, melanin, which is responsible for the color of skin. They predominantly occur in skin, but are also found in other parts of the body, including the bowel and the eye. Melanoma can occur in any part of the body that contains melanocytes.
  • Leukemia refers to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number of abnormal cells in the blood-leukemic or aleukemic (subleukemic).
  • Sarcoma is a cancer that arises from transformed connective tissue cells. These cells originate from embryonic mesoderm, or middle layer, which forms the bone, cartilage, and fat tissues. This is in contrast to carcinomas, which originate in the epithelium. The epithelium lines the surface of structures throughout the body, and is the origin of cancers in the breast, colon, and pancreas.
  • Myeloma as mentioned herein is a cancer of plasma cells, a type of white blood cell normally responsible for the production of antibodies. Collections of abnormal cells accumulate in bones, where they cause bone lesions, and in the bone marrow where they interfere with the production of normal blood cells. Most cases of myeloma also feature the production of a paraprotein, an abnormal antibody that can cause kidney problems and interferes with the production of normal antibodies leading to immunodeficiency. Hypercalcemia (high calcium levels) is often encountered.
  • Lymphoma is a cancer in the lymphatic cells of the immune system.
  • lymphomas present as a solid tumor of lymphoid cells. These malignant cells often originate in lymph nodes, presenting as an enlargement of the node (a tumor). It can also affect other organs in which case it is referred to as extranodal lymphoma.
  • Non limiting examples for lymphoma include Hodgkin's disease, non-Hodgkin's lymphomas and Burkitt's lymphoma.
  • malignancies that may find utility in the present invention can comprise but are not limited to hematological malignancies (including lymphoma, leukemia and myeloproliferative disorders, as described above), hypoplastic and aplastic anemia (both virally induced and idiopathic), myelodysplastic syndromes, all types of paraneoplastic syndromes (both immune mediated and idiopathic) and solid tumors (including GI tract, colon, lung, liver, breast, prostate, pancreas and Kaposi's sarcoma.
  • hematological malignancies including lymphoma, leukemia and myeloproliferative disorders, as described above
  • hypoplastic and aplastic anemia both virally induced and idiopathic
  • myelodysplastic syndromes all types of paraneoplastic syndromes (both immune mediated and idiopathic)
  • solid tumors including GI tract, colon, lung, liver, breast, prostate, pancreas and Kaposi's sarcoma
  • the invention may be applicable as well for the treatment or inhibition of solid tumors such as tumors in lip and oral cavity, pharynx, larynx, paranasal sinuses, major salivary glands, thyroid gland, esophagus, stomach, small intestine, colon, colorectum, anal canal, liver, gallbladder, extraliepatic bile ducts, ampulla of vater, exocrine pancreas, lung, pleural mesothelioma, bone, soft tissue sarcoma, carcinoma and malignant melanoma of the skin, breast, vulva, vagina, cervix uteri, corpus uteri, ovary, fallopian tube, gestational trophoblastic tumors, penis, prostate, testis, kidney, renal pelvis, ureter, urinary bladder, urethra, carcinoma of the eyelid, carcinoma of the conjunctiva, malignant melanoma of the conjunctiva, malignant
  • the method of the invention may be applicable in determining the efficacy and suitability of using different chemotherapeutic agents, and combinations thereof for treating colorectal cancer.
  • Colon cancer also referred to herein as "colorectal cancer or carcinoma" as herein defined is a disease in which malignant (cancer) cells form in the tissues of the colon (part of the body's digestive system).
  • Colorectal carcinoma is the third most common cancer in the United States after prostate and lung/bronchus cancers in men and after breast and lung/bronchus cancers in women. It is also the third leading cause of cancer- related death in the United States. In 2011, an estimated 141 ,210 new cases of colorectal carcinoma were diagnosed in United States, with an estimated 49,380 deaths.
  • Risk factors of colon cancer include age and health history, a family history of colon cancer, and in some cases having inflammatory bowel disease (IBD).
  • IBD inflammatory bowel disease
  • the prognosis of patients with colon cancer is clearly related to the degree of penetration of the tumor through the bowel wall, the presence or absence of nodal involvement, and the presence or absence of distant metastases, with these three characteristics forming the basis for all staging systems developed for this disease.
  • 5-fluorouracil has remained the mainstay of therapeutic options in the treatment of advanced CRC, with the introduction of newer agents such as oxaliplatin and irinotecan (CPT11) in combination with 5-FU increasing the response rates.
  • CPT11 irinotecan
  • monoclonal antibodies targeting the epidermal growth factor receptor or vascular endothelial growth factor has demonstrated additional clinical benefit for patients with metastatic disease.
  • the main monoclonal antibodies that are used for the treatment of colon cancer include bevacizumab (also known as Avastin), cetuximab (also known as Erbitux) and panitumumab (also known as Vectibix).
  • the method of the invention may be used for determining the appropriate treatment regimen for any type of colorectal cancer (CRC).
  • CRC colorectal cancer
  • Different types of colorectal cancers More specifically, collectively, collectively, collectively, collectively, collectively, collectively, colon cancer is defined as a cancer that forms in the tissues of the colon (the longest part of the large intestine). These tumors are sometimes referred to as "colorectal” cancer, reflecting the fact that the rectum, the end portion of the colon, may also be affected.
  • colorectal carcinomas More than 90% of colorectal carcinomas are adenocarcinomas, originating from epithelial cells of the colorectal mucosa. Other rare types of colorectal carcinomas include neuroendocrine, squamous cell, adenosquamous, spindle cell and undifferentiated carcinomas. Conventional adenocarcinoma is characterized by glandular formation, which is the basis for histologic tumor grading.
  • colon cancers occurs due to lifestyle and increasing age with only a minority of cases associated with underlying genetic disorders. It typically starts in the lining of the bowel and if left untreated, can grow into the muscle layers underneath, and then through the bowel wall.
  • the method of the invention may be used for determining the suitability for a specific treatment of a patient suffering from any autoimmune disease such as for example, but not limited to, inflammatory bowel disease (IBD), ulcerative colitis and Crohn's disease and fatty liver disease, rheumatoid arthritis, systemic lupus erythematosus (SLE), Eaton-Lambert syndrome, Goodpasture's syndrome, Greave's disease, Guillain-Barr syndrome, autoimmune hemolytic anemia (AIHA), hepatitis, insulin-dependent diabetes mellitus (IDDM) and NIDDM, multiple sclerosis (MS), myasthenia gravis, plexus disorders e.g.
  • IBD inflammatory bowel disease
  • SLE systemic lupus erythematosus
  • AIHA autoimmune hemolytic anemia
  • IDDM insulin-dependent diabetes mellitus
  • MS multiple sclerosis
  • myasthenia gravis plexus disorders e.g.
  • the method of the invention may be also applicable for determining the suitability for treatment of a patient suffering from inflammatory bowel diseases (IBD).
  • IBD inflammatory bowel diseases
  • Inflammatory bowel diseases are common gastrointestinal disorders, that can be perceived as being the result of a dysbalance between Thl -proinflammatory and Th2 -anti- inflammatory subtypes of immune responses.
  • IBD is a group of inflammatory conditions of the colon and small intestine.
  • the major types of IBD are Crohn's disease and ulcerative colitis (UC) that share the same symptoms such as diarrhea, vomiting, weight loss, fever and abdominal pain. Other forms of IBD account for far fewer cases.
  • Collagenous colitis Lymphocytic colitis
  • Ischaemic colitis Diversion colitis
  • Behcet's syndrome Indeterminate colitis which is an inability to make a definitive diagnosis distinguishing Crohn's disease from Ulcerative colitis.
  • the method of the invention may be used in determining the suitability for treatment of a patient suffering from rheumatoid arthritis.
  • Rheumatoid arthritis is a chronic, systemic autoimmune disorder that most commonly causes inflammation and tissue damage in joints (arthritis) and tendon sheaths, together with anemia. It can also produce diffuse inflammation in the lungs, pericardium, pleura, and the sclera of the eye, and also nodular lesions, most common in subcutaneous tissue. It can be a disabling and painful condition, which can lead to substantial loss of functioning and mobility.
  • the method of the invention may be used in determining the suitability for treatment of a patient suffering from a disease defined within the seronegative spondyloarthropathy category, which includes psoriatic arthritis, reactive arthritis, and ankylosing spondylitis, and is characterized by signs of inflammation, multiple joint involvement, and distal involvement in the hands and feet with added features of bone proliferation.
  • a disease defined within the seronegative spondyloarthropathy category which includes psoriatic arthritis, reactive arthritis, and ankylosing spondylitis, and is characterized by signs of inflammation, multiple joint involvement, and distal involvement in the hands and feet with added features of bone proliferation.
  • the method of the invention may be also applicable for determining the suitability of a specific therapeutic agent for treating a subject suffering from an infectious disease. More specifically, such infectious disease may be any one of protozoan diseases, viral diseases, bacterial diseases, parasitic diseases, fungal diseases and mycoplasma diseases. Therefore, specific embodiments of the invention relate to the use of the method of the invention for determining the suitability for treatment of a patient suffering from infectious diseases.
  • infectious disease as used herein also encompasses any infectious disease caused by a pathogenic agent.
  • Pathogenic agents include prokaryotic microorganisms, lower eukaryotic microorganisms, complex eukaryotic organisms, viruses, fungi, prions, parasites, yeasts, toxins and venoms.
  • a prokaryotic microorganism includes bacteria such as Gram positive, Gram negative and Gram variable bacteria and intracellular bacteria.
  • bacteria contemplated herein include the species of the genera Treponema sp., Borrelia sp., Neisseria sp., Legionelh sp., Bordetelh sp., Escherichia sp., Salmonella sp., Shigella sp., Klebsiella sp., Yersinia sp., Vibrio sp., Hemophilus sp., Rickettsia sp., Chlamydia sp., Mycoplasma sp., Staphylococcus sp., Streptococcus sp., Bacillus sp., Clostridium sp., Corynebacterium sp., Proprionibacterium sp., Mycobacterium sp., Ureaplasma
  • a lower eukaryotic organism includes a yeast or fungus such as but not limited to Pneumocystis carinii, Candida albicans, Aspergillus, Histoplasma capsulatum, Blastomyces dermatitidis, Cryptococcus neoformans, Trichophyton and Microsporum.
  • yeast or fungus such as but not limited to Pneumocystis carinii, Candida albicans, Aspergillus, Histoplasma capsulatum, Blastomyces dermatitidis, Cryptococcus neoformans, Trichophyton and Microsporum.
  • a complex eukaryotic organism includes worms, insects, arachnids, nematodes, aemobe, Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, Trypanosoma brucei gambiense, Trypanosoma cruzi, Bahntidium coli, Toxophsma gondii, Cryptosporidium or Leishmania.
  • viruses is used in its broadest sense to include viruses of the families adenoviruses, papovaviruses, herpesviruses: simplex, varicella-zoster, Epstein-Barr, CMV, pox viruses: smallpox, vaccinia, hepatitis B, rhinoviruses, hepatitis A, polio virus, rubella virus, hepatitis C, arboviruses, rabies virus, influenza viruses A and B, measles virus, mumps virus, HIV, HTLV I and II.
  • fungi includes for example, fungi that cause diseases such as ringworm, histoplasmosis, blastomycosis, aspergillosis, cryptococcosis, sporotrichosis, coccidioidomycosis, paracoccidio-idoinycosis, and candidiasis.
  • parasite includes, but not limited to, infections caused by somatic tapeworms, blood flukes, tissue roundworms, ameba, and Plasmodium, Trypanosoma, Leishmania, and Toxoplasma species.
  • chemotherapeutic agent or "chemotherapeutic drug” (also termed chemotherapy) as used herein refers to a drug treatment intended for eliminating or destructing (killing) cancer cells.
  • chemotherapeutic drugs also termed chemotherapy
  • the mechanism underlying the activity of some chemotherapeutic drugs is based on destructing rapidly dividing cells, as many cancer cells grow and multiply more rapidly than normal cells.
  • chemotherapeutic agents also harm cells that rapidly divide under normal circumstances, for example bone marrow cells, digestive tract cells, and hair follicles.
  • chemotherapeutic drugs are available.
  • a chemotherapeutic drug may be used alone or in combination with another chemotherapeutic drug or with other forms of cancer therapy, such as a biological drug, radiation therapy or surgery.
  • Certain chemotherapy agents have also been used in the treatment of conditions other than cancer, including ankylosing spondylitis, multiple sclerosis, Crohn's disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, lupus and scleroderma.
  • Chemotherapeutic drugs affect cell division or DNA synthesis and function and can be generally classified into groups, based on their structure or biological function.
  • the present invention generally pertains to chemotherapeutic agents that are classified as alkylating agents, anti-metabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other anti-tumor agents.
  • chemotherapeutic drugs may be classified as relating to more than a single group. It is noteworthy that some agents, including monoclonal antibodies and tyrosine kinase inhibitors, which are sometimes referred to as “chemotherapy”, do not directly interfere with DNA synthesis or cell division but rather function by targeting specific differences between cancer cells and normal cells and are generally referred to as “targeted therapies", “biological therapy” or “immunotherapeutic agent” as detailed below.
  • the invention provides a method for determining the efficacy of treating a subject suffering from a chronic inflammatory condition with a chemotherapeutic agent/s.
  • a chemotherapeutic agent may be at least one of an alkylating agent, an anti-metabolite, a plant alkaloid, a terpenoid, a taxane, an anthracycline, a topoisomerase inhibitor, a cytotoxic antibiotic and an anti-tumor agent.
  • the invention provides a method for determining the efficacy of treating a subject suffering from a chronic inflammatory condition with a combination of chemotherapeutic agent/s and at least one of biological agent and immuno-therapeutic agent.
  • the invention provides a method for determining the efficacy of treating a subject suffering from a chronic inflammatory condition with a chemotherapeutic agent that may be at least one alkylating agent.
  • alkylating agents function by alkylating many nucleophilic functional groups under conditions present in cells.
  • chemotherapeutic agents that are considered as alkylating agents are cisplatin and carboplatin, as well as oxaliplatin.
  • Alkylating agents impair cell function by forming covalent bonds with amino, carboxyl, sulfhydryl, and phosphate groups in various biologically-significant molecules.
  • agents which function by chemically modifying DNA are mechlorethamine, cyclophosphamide, chlorambucil and ifosfamide.
  • An additional agent acting as a cell cycle non-specific alkylating antineoplastic agent is the alkyl sulfonate agent busulfan (also known as Busulfex).
  • the method of the invention may be used to determine the effect of alkylating chemotherapeutic agents on a host's immune system.
  • the method of the invention using the expression level of CD247 as a biomarker is exemplified herein below in Example 1 for the alkylating chemo therapeutic agent cyclophosphamide.
  • the invention provides a method for determining the efficacy of treating a chronic inflammatory condition, specifically, a proliferative disorder, with Cyclophosphamide.
  • the chemotherapeutic agent Cyclophosphamide also known by the trade names Endoxan, Endoxan, Cytoxan, Neosar, Procytox and Revimmune or by the name cytophosphane
  • Cyclophosphamide is a nitrogen mustard alkylating agent of the oxazophorines group that acts by adding an alkyl group to DNA (particularly to guanine, at the number 7 nitrogen atom of the imidazole ring).
  • Cyclophosphamide is used alone for the treatment of several types of cancers but often in combination with other drugs to treat breast cancer, leukemia and ovarian cancer. It also is approved for treating nephrotic syndrome (a disease of the kidneys) in children. Unapproved uses include the treatment of Wegener's granulomatosis, severe rheumatoid arthritis, lupus erythematosus, advanced mycosis fungoides, and several of forms of vasculitis.
  • the method of the invention may be used to determine the efficacy of the alkylating chemotherapeutic agent busulfan on a host's immune system.
  • the invention provides a method for determining the efficacy of treating a chronic inflammatory condition, specifically, a proliferative disorder, with Busulfan.
  • the chemotherapeutic agent Busulfan also known by the trade names Myleran and Busulfex, or by the chemical designation is 1 ,4-butanediol dimethanesulfonate
  • Busulfan is a cell cycle non-specific alkylating anti-neoplastic agent, in the class of alkyl sulfonates. Its mechanism of action through alkylation produces guanine-adenine intrastrand crosslinks. This type of damage to the DNA cannot be repaired by cellular machinery and thus the cell undergoes apoptosis. Busulfan is given in high doses before a stem cell transplant to treat some types of leukemia and other types of cancer. It may also be given in standard doses to treat other blood-related disorders.
  • the invention provides a method for determining the efficacy of treating a subject suffering from a chronic inflammatory condition with a chemotherapeutic agent that may be at least one Anti-metabolite.
  • Anti-metabolites also termed purine and pyrimidine analogues
  • the incorporation of anti-metabolites into DNA interferes with DNA syntheses, leading to abnormal cell development and division.
  • Anti-metabolites also affect RNA synthesis.
  • anti-metabolites include 5-fiuorouracil (5-FU), azathioprine and mercaptopurine, fludarabine, cladribine (2-chlorodeoxyadenosine, 2-CdA), pentostatin (2'-deoxycoformycin, 2'-DCF), nelarabine, Floxuridine (FUDR) and Cytosine arabinoside (Cytarabine).
  • 5-fiuorouracil 5-FU
  • azathioprine and mercaptopurine fludarabine
  • cladribine (2-chlorodeoxyadenosine, 2-CdA
  • pentostatin (2'-deoxycoformycin
  • 2'-DCF pentostatin
  • nelarabine Floxuridine
  • Cytosine arabinoside Cytarabine
  • the present invention demonstrates determining the expression level of CD247 for assessing the efficacy of treatment with anti-metabolite chemotherapeutic agents during chronic inflammation, particularly, the anti-metabolite agent 5-fluorouracil (5-FU).
  • anti-metabolite agent 5-fluorouracil 5-fluorouracil
  • the method of the invention may be applicable for determining the efficacy of treatment of a subject suffering from a chronic inflammatory condition with 5-FU.
  • the chemotherapeutic drug 5-Fluorouracil also known as Fluorouracil, 5-FU or f5U, or by the trade names Adrucil, Carac, Efudix, Efudex and Fluoroplex
  • Adrucil, Carac, Efudix, Efudex and Fluoroplex is a pyrimidine analogue anti-metabolite which is used in the treatment of cancer, through the irreversible inhibition of thymidylate synthase.
  • 5-FU is mainly used in the treatment of colorectal cancer and pancreatic cancer and is sometimes used in the treatment of inflammatory breast cancer.
  • the invention provides a method for determining the efficacy of treating a subject suffering from a chronic inflammatory condition with a chemotherapeutic agent that may be at least one Plant alkaloid and terpenoid.
  • Plant alkaloids and terpenoids are agents derived from plants that block cell division by preventing microtubule function, thereby inhibiting the process of cell division (also known as "mitotic inhibitors" or "anti-mitotic agents").
  • Examples of alkaloids include the vinca alkaloids (e.g. vincristine, vinblastine, vinorelbine and vindesine) and terpenoids include, for example, taxanes (e.g. paclitaxel and docetaxel). Taxanes act by enhancing the stability of microtubules, preventing the separation of chromosomes during anaphase.
  • the invention provides a method for determining the efficacy of treating a subject suffering from a chronic inflammatory condition with a chemotherapeutic agent that may be at least one Topoisomerase inhibitor.
  • Topoisomerases are essential enzymes that maintain DNA topology (i.e. the overall three dimensional structure of DNA). Inhibition of type I or type II topoisomerases interferes with both transcription and replication of DNA by inhibiting proper DNA supercoiling.
  • Type I topoisomerase inhibitors include camptothecins (e.g. irinotecan and topotecan) and examples of type II inhibitors include amsacrine, etoposide, etoposide phosphate, and teniposide.
  • the present invention provides a method for determining the efficacy of a treatment by type I topoisomerase inhibitors as a therapeutic agent during a state of chronic inflammation, by determining the expression level of CD247.
  • the present invention exemplifies the effect on the host immune state in response to a treatment with the topoisomerase inhibitor irinotecan (CPTl l) alone (Example 1 below) or in combination with the anti-metabolite agent 5-FU (Examples 8 and 9).
  • the invention provides a method for determining the efficacy of treating a chronic inflammatory condition, specifically, a proliferative disorder, with Irinotecan.
  • a chronic inflammatory condition specifically, a proliferative disorder
  • Irinotecan chemotherapeutic agent
  • the chemotherapeutic agent Irinotecan also known as CPT-11
  • CPT-11 which is a semisynthetic analogue of the natural alkaloid camptothecin
  • Irinotecan acts by preventing DNA from unwinding, via the inhibition of topoisomerase I. It is mainly used for the treatment of colon cancer, in particular, in combination with other chemotherapy agents (including the regimen FOLFIRI, which consists of infusional 5-fluorouracil, leucovorin, and irinotecan.
  • the invention provides a method for determining the efficacy of treating a subject suffering from a chronic inflammatory condition with a chemotherapeutic agent that may be at least one Anthracycline.
  • Anthracyclines are a class of drugs used in cancer chemotherapy that are derived from the streptomyces bacterium. These compounds are used to treat many cancers, including leukemias, lymphomas, breast, uterine, ovarian, and lung cancers. These agents include, inter alia, the drugs daunorubicin (also known as Daunomycin), and doxorubicin and many other related agents (e.g., Valrubicin and Idarubicin). For example, the anthracycline agent Idarubicin acts by interfering with the enzyme topoisomerase II.
  • the present invention provides a method for determining the efficacy of a treatment by anthracycline chemotherapeutic agents, particularly by doxorubicin, during a state of chronic inflammation, by determining the expression level of CD247.
  • the invention provides a method for determining the efficacy of treating a chronic inflammatory condition, specifically, a proliferative disorder, with Doxorubicin.
  • the chemotherapeutic agent Doxorubicin also known by the trade name Adriamycin and by the name hydro xydaunorubicin
  • Doxorubicin is an anthracycline antibiotic that is closely related to the natural product daunomycin, and like all anthracyclines, it works by intercalating DNA.
  • the most serious adverse side effect of using this agent is the life-threatening heart damage. It is commonly used in the treatment of a wide range of cancers, including hematological malignancies, many types of carcinoma, and soft tissue sarcomas.
  • the invention provides a method for determining the efficacy of treating a subject suffering from a chronic inflammatory condition with a chemotherapeutic agent that may be at least one Cytotoxic antibiotics.
  • a chemotherapeutic agent that may be at least one Cytotoxic antibiotics.
  • the anthracyclines agents described above are also classified as "cytotoxic antibiotics".
  • Cytotoxic antibiotics also include the agent actinomycin D (also known generically as Actinomycin or Dactinomycin), which is the most significant member of the actinomycines class of polypeptide antibiotics (that were also isolated from streptomyces). Actinomycin D is shown to have the ability to inhibit transcription by binding DNA at the transcription initiation complex and preventing elongation of RNA chain by RNA polymerase.
  • Other cytotoxic antibiotics include bleomycin and mitomycin.
  • the method of the invention as well as any compositions and kits described herein after are applicable in determining the efficacy of treatment and monitoring a disease progression in response to treatment with any of the chemotherapeutic agents, any combinations of at least two chemotherapeutic agents, any combination with a biological agent or any immuno-therapeutic agents or any combinations thereof.
  • combination with refers to administration or two or more therapies over the course of a treatment regimen, where the therapies may be administered together or separately, and, where used in reference to drugs, may be administered in the same or different formulations, by the same or different routes, and in the same or different dosage form type.
  • the present invention thus provides an efficient method for determining the efficacy and suitability of a certain therapeutic agent, specifically, an immuno-therapeutic agent or any combinations thereof with other chemontherapeutic agents.
  • immunotherapy is a treatment that uses certain parts of the immune system to fight diseases (e.g. cancer), by, inter alia, stimulating the immune system to become more efficient in attacking cancer cells (e.g., by administering vaccines) or by administering components of the immune system (e.g., by administering cytokines, antibodies, etc.).
  • immunotherapy has become an important part of treating several types of cancer with the main types of immunotherapy used being monoclonal antibodies (either naked or conjugated), cancer vaccines (i.e. that induce the immune system to mount an attack against cancer cells in the body) and non-specific immunotherapies.
  • the immune-therapeutic agent may be at least one of adoptive cell transfer, a cancer vaccine, antibody-based therapy, a hormone, a cytokine or any combination thereof.
  • cancer vaccines as referred to herein are vaccines that induce the immune system to mount an attack against cancer cells in the body.
  • a cancer treatment vaccine uses cancer cells, parts of cells, or pure antigens to increase the immune response against cancer cells that are already in the body. These cancer vaccines are often combined with other substances or adjuvants that enhance the immune response.
  • Non-specific immunotherapies as referred to herein do not target a certain cell or antigen, but rather stimulate the immune system in a general way, which may still result in an enhanced activity of the immune system against cancer cells.
  • a non-limiting example of non-specific immunotherapies includes cytokines (e.g. interleukins, interferons).
  • Antibody-mediated therapy refers to the use of antibodies that are specific to a cancer cell or to any protein derived there-from for the treatment of cancer.
  • such antibodies may be monoclonal or polyclonal which may be naked or conjugated to another molecule.
  • Antibodies used for the treatment of cancer may be conjugated to a cytotoxic moiety or radioactive isotope, to selectively eliminate cancer cells.
  • adoptive transfer applies to all the therapies that consist of the transfer of components of the immune system that are already capable of mounting a specific immune response.
  • Examples of adoptive transfer include both the transfer of antibodies and also, specific types of cells that are capable of mediating antigen-specific tumor regression such as LAK and T cells.
  • Cell-based therapies with various lymphocytes and antigen-presenting cells are promising approaches for cancer immunotherapy.
  • the transfusion of T lymphocytes also called adoptive cell therapy (ACT) is an effective treatment for viral infections and has induced regression of cancer in early stage clinical trials.
  • CD247 expression may serve as a sensitive biomarker sensing whether treatment with a certain immuno -therapeutic agent such as the TNF-a inhibitor, Etanercept, may be effective in reducing or eliminating the chronic inflammatory condition.
  • a certain immuno -therapeutic agent such as the TNF-a inhibitor, Etanercept
  • Etanercept also known by the trade name Enbrel
  • TNF tumor necrosis factor
  • Etanercept is a large molecule, with a molecular weight of 150 kDa, that binds to TNFa and decreases its role in disorders involving excess inflammation in humans and other animals, including autoimmune diseases such as ankylosing spondylitis, juvenile rheumatoid arthritis, psoriasis, psoriasis arthritis, rheumatoid arthritis and, potentially, in a variety of other disorders mediated by an excess of TNFa, the therapeutic potential being based on the fact that TNFa is the "master regulator" of the inflammatory response in many organ systems.
  • biological treatment refers to any biological material that affects different cellular pathways.
  • agent may include antibodies, for example, antibodies directed to cell surface receptors participating in signaling, that may either activate or inhibit the target receptor.
  • biological agent may also include any soluble receptor, cytokine, peptides or ligands.
  • monoclonal antibodies that are used for the treatment of cancer include bevacizumab (also known as Avastin), cetuximab (also known as Erbitux), anti-CTLA4 anibody and panitumumab (also known as Vectibix) and anti Grl antibodies.
  • the biological sample tested by the method of the invention may be any one of a blood sample, a spleen biopsy, cells from lymph nodes and a tissue biopsy, specifically, colon tissue.
  • sample in the present specification and claims is meant to include biological samples.
  • Biological samples may be obtained from mammal, specifically, a human subject, include fluid, solid (e.g., stool) or tissues.
  • sample may also include body fluids such as whole blood sample, blood cells, bone marrow, lymph fluid, serum, plasma, urine, sputum, saliva, faeces, semen, spinal fluid or CSF, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, milk, any human organ or tissue, any biopsy, for example, lymph node or spleen biopsies, any sample taken from any tissue or tissue extract, any sample obtained by lavage optionally of the breast ductal system, plural effusion, samples of in vitro or ex vivo cell culture and cell culture constituents.
  • body fluids such as whole blood sample, blood cells, bone marrow, lymph fluid, serum, plasma, urine, sputum, saliva, faeces, semen, spinal fluid or CSF, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, milk, any human organ or tissue, any biopsy, for example, lymph node or spleen biopsies, any sample
  • Biological samples may be obtained from all of the various families of domestic animals, as well as feral or wild animals, including, but not limited to, such animals as ungulates, bear, fish, lagamorphs, rodents, etc.
  • the sample is liquid, specifically, a body fluid sample, most preferably, a serum sample and of mammalian origin, specifically, human.
  • a blood sample is being used by the invention.
  • the method of the invention is applicable for testing any tissue sample, for example, lymph nodes and spleen, biopsies.
  • the present invention relates to the diagnosis and monitoring of subjects or patients, in need thereof.
  • patient or “subject in need” it is meant any organism who may be affected by the above-mentioned conditions, and to whom the monitoring and diagnosis methods herein described is desired, including humans, domestic and non-domestic mammals such as canine and feline subjects, bovine, simian, equine and murine subjects, rodents, domestic birds, aquaculture, fish and exotic aquarium fish. It should be appreciated that the diagnosed or monitored subject may be also any reptile or zoo animal. More specifically, the methods of the invention are intended for mammals.
  • mammalian subject is meant any mammal for which the proposed therapy is desired, including human, livestock, equine, canine, and feline subjects, most specifically humans.
  • the invention relates to a composition
  • a composition comprising:
  • detecting molecules of (a) and (b) may be attached to a solid support.
  • the composition of the invention may be for use in a method for determining the efficacy of a treatment with a therapeutic agent on a subject suffering from a chronic inflammatory condition. More specifically, such method provides determining whether a subject suffering from a chronic inflammatory condition would respond, specifically, in exhibiting a beneficial response to a treatment with a therapeutic agent.
  • such therapeutic agent may be at least one chemotherapeutic agent, at least one immunotherapeutic agent or any combination thereof.
  • the detecting molecules specific for CD247 comprised within the composition of the invention may be selected from isolated detecting amino acid molecules and isolated detecting nucleic acid molecules.
  • such detecting amino acid molecule specific for CD247 may be an isolated antibody that specifically recognizes and binds CD247.
  • compositions of the invention may further comprise detecting molecules specific for control reference protein.
  • control reference protein may be used for normalizing the detected expression levels for the biomarker of the invention CD247.
  • composition of the invention comprises at least one reference control that may be at least one of CD3s, CD38, CD3y, TCRa, TCRP and CD56.
  • the composition of the invention may be used for determining the efficacy of a therapeutic agent that may be a chemotherapeutic agent that may be at least one of an alkylating agent, an anti-metabolite, a plant alkaloid, a terpenoid, a taxane, an anthracycline, a topoisomerase inhibitor, a cytotoxic antibiotic and an anti-tumor agent.
  • a therapeutic agent that may be a chemotherapeutic agent that may be at least one of an alkylating agent, an anti-metabolite, a plant alkaloid, a terpenoid, a taxane, an anthracycline, a topoisomerase inhibitor, a cytotoxic antibiotic and an anti-tumor agent.
  • composition of the invention may be used in a method for determining the efficacy of treatment with an immune-therapeutic agent that may be at least one of adoptive cell transfer, a cancer vaccine, antibody-based therapy, a hormone, a cytokine or any combination thereof.
  • an immune-therapeutic agent may be at least one of adoptive cell transfer, a cancer vaccine, antibody-based therapy, a hormone, a cytokine or any combination thereof.
  • the compositions described by the invention or any components thereof, specifically, the detecting molecules may be attached to a solid support.
  • the solid support may include polymers, such as polystyrene, agarose, sepharose, cellulose, glass, glass beads and magnetizable particles of cellulose or other polymers.
  • the solid-support can be in the form of large or small beads, chips or particles, tubes, plates, or other forms.
  • diagnostic and prognostic agents of the present invention can be packaged in a diagnostic kit.
  • diagnostic kits can include an antibody (e.g., labeled) of the present invention in one container and a solid phase for attaching multiple biological samples packaged in a second container as well as imaging reagent in a third container (e.g., secondary labeled antibody) with appropriate buffers and preservatives and used for diagnosis.
  • an antibody e.g., labeled
  • imaging reagent e.g., secondary labeled antibody
  • kits comprising:
  • the kit of the invention may include at least one control sample.
  • the control sample may include samples of subjects (preferably, with a matching gender and age) suffering from a specific chronic inflammatory condition, samples from treated subjects that display a beneficial response (“responders"), samples of treated subjects that do not respond (non responders), samples from healthy subjects, and optionally, samples of subjects suffering from a different pathologic condition.
  • the kit of the invention used in a method for determining the efficacy of a treatment with a therapeutic agent on a subject suffering from a chronic inflammatory condition. More specifically, the kit of the invention may be used for determining whether a subject suffering from a chronic inflammatory condition would respond and would exhibit a beneficial response to a treatment with a therapeutic agent.
  • Such therapeutic agent may be at least one chemotherapeutic agent, at least one immunotherapeutic agent or any combination thereof.
  • kit of the invention may be applicable for monitoring and assessing responsiveness of a subject suffering from a chronic-inflammatory condition to a treatment with a therapeutic agent.
  • Such instructions may include at least one of:
  • instructions for carrying out the determining of the level of expression of CD247 in a biological sample and optionally for determining the expression level of at least one reference control More specifically, instructions for carrying out the detection and quantification of expression of the biomarker of the invention, CD247 and of at least one said control reference in the tested sample. Such instruction may also indicate procedure for obtaining an expression value of said CD247 in said sample.
  • the kit of the invention may further comprise instructions for normalizing the expression levels of CD247 in a sample as compared to a reference control, for example, CD3s or any one of CD38, CD3y, TCRa, TCRJ3 and CD56. Still further, in specific embodiments, where an active calibration curve is being used, the kit of the invention may further comprise any reagents necessary for preparing such calibration curve. It should be understood that such active calibration curve serves as a reference curve adjusted for each particular method used for determining the expression of CD247 in the examined sample. In one particular example, the kit of the invention may include beads/or fixed cells stained with different fluorescent intensities representing the range of the CD247 expression levels in the healthy population of age and gender matched subjects.
  • a positive rate of change of said expression values in a sample obtained after initiation of said treatment as compared to the CD247 expression value in a sample obtained prior to initiation of said treatment is indicative of the responsiveness of said subject to said treatment, that leads either directly or indirectly to an anti-inflammatory effect.
  • the kit of the invention may comprise at least one calibration curve.
  • Such calibration curves may include at least one of (a) a pre-determined calibration curve providing normalized standard expression values of said CD247 (b) a predetermined standard [cutoff] rate of change between expression values of CD247 obtained for at least one subject prior to said therapy and after the initiation of said therapy; (c) a predetermined standard [cut-off] rate of change between expression values of CD247 obtained for at least one subject in at least two temporally- separated samples obtained for said at least one subject after the initiation of therapy.
  • the kit of the invention comprises detecting molecules specific for CD247 and optionally detecting molecules specific for at least one reference control.
  • detecting molecules may be selected from isolated detecting amino acid molecules and isolated detecting nucleic acid molecules.
  • the detecting amino acid molecule specific for CD247 may be an isolated antibody that specifically recognizes and binds CD247.
  • the kit of the invention may further comprise detecting molecules specific for reference control that may be at least one of CD3s, CD33 ⁇ 4 CD3y, TCRa, TCRP, CD56 and in some embodiments also TCRy or TCR ⁇ .
  • the kit of the invention may be particularly suitable for determining if a specific chemotherapeutic agent exhibits a beneficial effect on a subject suffering from a chronic inflammatory condition.
  • chemotherapeutic agent may be at least one of an alkylating agent, an anti-metabolite, a plant alkaloid, a terpenoid, a taxane, an anthracycline, a topoisomerase inhibitor, a cytotoxic antibiotic and an anti-tumor agent.
  • the kit of the invention may be used for determining the suitability of using an immuno therapeutic agent for the treatment of a chronic inflammatory condition.
  • an immunotherapeutic agent may include at least one of adoptive cell transfer, a cancer vaccine, antibody-based therapy, a hormone, a cytokine or any combination thereof.
  • the kit of the invention may be specifically useful in determining an optimal treatment for a subject suffering from any chronic inflammatory condition, for example, a proliferative disorder, an autoimmune disorder or an infectious disorder. More specific embodiments relate to a proliferative disorder.
  • the kit of the invention may further comprise at least one of: (a) , detecting molecules specific for determining the level of expression of S100A8 and/or S100A9 in a biological sample;
  • kits of the invention may further comprise reagents required for performing said assays.
  • the kit of the invention may further comprise detecting amino acid molecules such as any one of isolated antibody that specifically recognizes and binds CDl lb and an isolated antibody that specifically recognizes and binds Grl, antibodies recognizing the S100A8 and S100A9 proteins etc.
  • the detecting molecules for CD247 expression are isolated detecting nucleic acid molecules.
  • such detecting nucleic acid molecules may be isolated oligonucleotides, each oligonucleotide specifically hybridizes to a nucleic acid sequence of the RNA products of said CD247.
  • the oligonucleotide used as a detecting molecule according to certain embodiments of the invention may be any one of a pair of primers or nucleotide probe.
  • the level of expression of CD247 may be determined using a nucleic acid amplification assay selected from the group consisting of: a Real-Time PCR, micro arrays, PCR, in situ Hybridization and Comparative Genomic Hybridization.
  • a nucleic acid based detecting molecule is an aptamer
  • the detection of CD247 expression is performed at the protein level.
  • the kit of the invention may comprise detecting molecules for at least one reference control protein.
  • reference control may be any one of CD3s, CO38, CD3y, TCRa and , TCRp, for T cells, CD56 and SNX27.
  • the kit of the invention may comprise detecting molecules specific for CD3s that is used as the reference control protein.
  • CD56 is used as a reference control.
  • anther embodiment of the invention relates to SNX27 as a control reference.
  • the kit of the invention may be adapted for examining different biological samples.
  • the biological sample may be any one of a whole blood sample, blood cells, bone marrow, lymph fluid, Spleen lymph nodes tissue samples, serum, plasma, urine, sputum, saliva, faeces, semen, spinal fluid or CSF, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, milk, any human organ or tissue, any sample obtained by lavage optionally of the breast ductal system, plural effusion, samples of in vitro or ex vivo cell culture and cell culture constituents.
  • the biological sample may be a blood sample.
  • the kit of the invention may therefore optionally comprise suitable mans for obtaining said sample. More specifically, for using the kit of the invention, one must first obtain samples from the tested subjects. To do so, means for obtaining such samples may be required.
  • Such means for obtaining a sample from the mammalian subject can be any means for obtaining a sample from the subject known in the art. Examples for obtaining e.g. blood or bone marrow samples are known in the art and could be any kind of finger or skin prick or lancet based device, which basically pierces the skin and results in a drop of blood being released from the skin.
  • aspirating or biopsy needles may be also used for obtaining spleen lymph nodes tissue samples. Samples may of course be taken from any other living tissue, or body secretions comprising viable cells, such as biopsies, saliva or even urine.
  • detecting molecules used for detecting the expression levels of CD247 may be provided in a kit attached to an array.
  • a "detecting molecule array” refers to a plurality of detection molecules that may be nucleic acids based or protein based detecting molecules (specifically, antibodies), optionally attached to a support where each of the detecting molecules is attached to a support in a unique pre- selected and defined region.
  • an array may contain different detecting molecules, such as specific antibodies or primers. It should be noted that each detecting molecule may be spatially arranged in a predetermined and separated location in an array.
  • an array may be a plurality of vessels (test tubes), plates, micro-wells in a micro-plate, each containing different detecting molecules, specifically, antibodies, against CD247.
  • An array may also be any solid support holding in distinct regions (dots, lines, columns) different and known, predetermined detecting molecules.
  • solid support is defined as any surface to which molecules may be attached through either covalent or non-covalent bonds.
  • useful solid supports include solid and semi-solid matrixes, such as aerogels and hydrogels, resins, beads, biochips (including thin film coated biochips), microfiuidic chip, a silicon chip, multi- well plates (also referred to as microtiter plates or microplates), membranes, filters, conducting and nonconducting metals, glass (including microscope slides) and magnetic supports.
  • useful solid supports include silica gels, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, polysaccharides such as Sepharose, nylon, latex bead, magnetic bead, paramagnetic bead, superparamagnetic bead, starch and the like.
  • This also includes, but is not limited to, microsphere particles such as Lumavidin.TM. or LS-beads, magnetic beads, charged paper, Langmuir-Bodgett films, functionalized glass, germanium, silicon, PTFE, polystyrene, gallium arsenide, gold, and silver.
  • any of the reagents, substances or ingredients included in any of the methods and kits of the invention may be provided as reagents embedded, linked, connected, attached, placed or fused to any of the solid support materials described above.
  • the diagnostic kits and methods of the invention further provide a tool for a "tailor- made” or personalized therapy, by identifying subjects suffering from a specific chronic inflammation that are likely to be benefit from treatment with an anti-inflammatory therapeutic agent.
  • a method of selecting a treatment regimen for treating a subject diagnosed with a chronic inflammatory condition comprising: (a) determining and evaluating the efficacy of a treatment with a therapeutic agent given to a subject suffering from a chronic-inflammatory condition according to the method of some embodiments of the invention, and (b) selecting a treatment regimen based on the evaluation; thereby selecting the treatment regimen for treating the subject diagnosed with said chronic inflammatory condition.
  • the invention provides a method of treating of a subject diagnosed with a chronic inflammatory condition, by evaluating the efficacy of a treatment, specifically, either a direct or indirect anti-inflammatory treatment, and selecting a treatment regimen based on the evaluation.
  • treatment or prevention refers to the complete range of therapeutically positive effects of administrating to a subject including inhibition, reduction of, alleviation of, and relief from, a chronic inflammatory condition and illness, chronic inflammation symptoms or undesired side effects or chronic inflammatory related disorders. More specifically, treatment or prevention of relapse re recurrence of the disease in response to a treatment with a non-effective, or deleterious therapeutic agent, includes the prevention or postponement of development of the disease, prevention or postponement of development of symptoms and/or a reduction in the severity of such symptoms that will or are expected to develop. These further include ameliorating existing symptoms, preventing- additional symptoms and ameliorating or preventing the underlying metabolic causes of symptoms.
  • the terms “inhibition”, “moderation”, “reduction” or “attenuation” as referred to herein, relate to the retardation, restraining or reduction of a process by any one of about 1 % to 99.9%, specifically, about 1 % to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%.
  • percentage values such as, for example, 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change” values, i.e., 0.1 , 0.5, 1.2, 5, etc., respectively.
  • the terms “decrease”, “inhibition”, “moderation” or “attenuation” as referred to herein, relate to the retardation, restraining or reduction of CD247 expression or levels by any one of about 1 % to 99.9%, specifically, about 1 % to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%.
  • the terms “increase”, “elevation”, “enhancement” or “elevation” as referred to herein, relate to the enhancement and increase of CD247 expression or levels by any one of about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%.
  • compositions comprising, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.
  • consisting essentially of means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • compositions comprising, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”. This term encompasses the terms “consisting of” and “consisting essentially of”.
  • Consisting essentially of means that the composition or method may include additional ingredients and/or steps, but only if the additional ingredients and/or steps do not materially alter the basic and novel characteristics of the claimed composition or method.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • the phrases "ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.
  • the term "method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • mice female, 6-8 weeks old mice were purchased from Harlan and were grown at the Hebrew University specific-pathogen-free facility. All experiments were done in accordance with pre-approved institutional protocols.
  • mice Induced chronic inflammation in mice was established by sustained subcutaneous injections of the heat-killed BaciUe Calmette Guerin strain of Mycobacterium tuberculosis, (231141 ; Difco Laboratories) (BCG, 50 ⁇ g per animal/dose) once a week, for three weeks.
  • BCG BaciUe Calmette Guerin strain of Mycobacterium tuberculosis
  • the first two BCG administrations were mixed 1 : 1 with Incomplete Freund's adjuvant (IF A) (Sigma), and the second boost delivery was given with PBS.
  • Mice sacrifice and cell isolations were performed two days after the third injection. Spleen weight was measured and cells from the spleen and peripheral blood (PBLs) were analyzed. The results represent three independent experiments.
  • CRC colorectal cancer
  • AOM azoxymethan
  • DSS DSS
  • PBLs peripheral blood cells
  • mice were randomly divided into two groups of four mice per group, as follows: (a) chronically inflamed control group, and (b) chronically inflamed treated group.
  • Chemotherapy treatment was given intraperitoneal (i.p) or intravenous (i.v.) once at day 7 or twice at day 7 and 14, using the following FDA-approved drugs at the indicated dosages: Mitomycin (2.5mg/kg, 3.25mg/kg, 4mg/kg, and 8mg/kg), Irinotecan (CPT11) (50mg/kg), Busulfan (lOmg/kg), Doxorubicin (Rubex) (8mg/kg), and Cyclophosphamide (lmg or 2.5mg per animal/dose). Each experiment also included a group of 4 naive mice.
  • the TNFa antagonist etanercept (Wyeth, UK) was used, which is a soluble form of the TNF receptor, comprising the Fc portion of IgGl and the extracellular domain of the TNF receptor (p75) [31]
  • Etanercept was administered daily (0.5mg/dose) by systemic intraperitoneal injection, starting from 1 day before the second BCG injection, until one day before cell harvest (Figure 6A). Control mice were injected with PBS.
  • Isolated splenocytes and peripheral blood leukocytes were treated with erythrocyte lysis buffer and washed in PBS.
  • Cells were pre-coated with anti- CD 16/CD32 (Bio legend) for FcR blockage.
  • FcR blockage For surface staining, cells were incubated for 30 min at 4°C with FITC-labeled anti-Grl, anti-CD 11c, and anti-CD4; PE-labeled anti F4/80, anti-CD3s, anti-CD45R/B220, and anti-mNKp46; and biotinylated anti- CD l ib and anti-CD8 that were detected with streptavidin-Cy5 (Biolegend and eBioscience).
  • Intracellular staining of CD247 cells was performed by subjecting the cells to fixation with paraformaldehyde (1%) for 20 min at 4°C, followed by washing and permeabilization for 10 min with saponin detergent (0.1%) at room temperature (RT). Cells were then washed and incubated with FITC-labeled monoclonal anti-CD247 (H146) (Abeam), or biotinylated monoclonal anti-CD247 (H146), which were subsequently detected with streptavidin-Cy5. FoxP3 staining was performed according to the manufacturer's instructions. Samples were analyzed using FACSCalibur with Cell Quest software (BD).
  • Analyses used either the splenocyte population or peripheral blood as mentioned above.
  • Cells (5 xl0 7 /ml, or 1 xl0 6 /ml) were lysed with Tris-NaCl buffer containing 0.5% Triton X-100 and protease inhibitors for 30 min on ice. Proteins were resolved on 12% SDS-PAGE and subjected to immunoblotting analysis using the following specific antibodies: anti-CD247, antiCD3s antibodies, anti-S100A9, anti-S100A8, anti-iNOS, anti-ARGl , and anti-aTubulin antibodies.
  • NK-cells The in-vivo cytotoxic activity of NK-cells was determined using donor splenocytes from BALB/c and C57BL/6 mice that were stained with carboxyfluorescein succinimidyl ester (CFSE), at 0.5 ⁇ CFSE low and at 5 ⁇ CFSE ⁇ for 10 min at 37°C.
  • Cells (5x10 10 ) of each type were mixed and injected into the tail vain of recipient C57BL/6 mice. After the transfer (24 hours) mice were sacrificed and splenocytes and PBLs were harvested.
  • the ratio between the syngeneic cells (CFSE 1 ⁇ 11 ) and alio geneic cells (CFSE l0W ) was evaluated using FACS analysis.
  • CD247 is a biomarker sensing the effect of chemotherapeutic drugs on the hosts' immune system
  • chemotherapeutic drugs such as for example Irinotecan (CPTl l), Cycophosphamide, Doxorubicin (Rubex), Busulfan and 5- fluorouracil (5FU), operate via different pathways (e.g. certain drugs are directed at a specific or non-specific stage of the cell cycle, while other drugs act by inducing DNA damage, etc.), all aimed at affecting tumor cell growth.
  • CPTl l Irinotecan
  • Cycophosphamide Cycophosphamide
  • Doxorubicin (Rubex)
  • Busulfan 5- fluorouracil
  • 5- fluorouracil 5- fluorouracil
  • the results presented herein demonstrate the effect of different chemotherapeutic drugs on the host's immune functional status. Specifically, the inventors demonstrated the unique effects of the various drugs on the host's immune status by following the protein CD247 as a biomarker.
  • chemotherapeutic drugs are considered as "antagonistic" to the hosts' immune system, i.e., these drugs act to enhance the host's immunosuppression.
  • these chemotherapeutic drugs are irinotecan and cyclophosphamide, the effect of which on the immunosuppressive state of the host's immune system was examined as described herein after.
  • the first chemotherapeutic drug that was used for the assessment of CD247 as a biomarker was Irinotecan (CPTl l), which is known to be applied for the treatment of metastatic colorectal cancer [27-28], ovarian cancer [29] and sometimes of lung cancer [30], where all these cancer types are characterized by chronic inflammatory microenvironment.
  • Inflamed mice i.e. mice induced with chronic inflammation, as described above that exhibited immunosuppressive features similar to those observed during tumor growth
  • CPTl l either by i.v. or by i.p. treatment, as described in Experimental procedures.
  • CPTl l either administered i.p. or i.v. significantly enhanced myelo id-derived suppressor cells (MDSC) levels in the spleen of the inflamed mice ( Figure 1A, left and right panels, respectively)
  • MDSC myelo id-derived suppressor cells
  • Treg cells a subpopulation of T cells that modulate the immune system, also known as "Regulatory T cells”, or “suppressor T cells”
  • Regulatory T cells or “suppressor T cells”
  • NK cell cytotoxic activity was affected as well, as indicated by the impaired in vivo killing activity measured by the clearance rate of allogeneic cells (Figure 2D), which correlated with low CD247 expression in NK (NCR+) cells ( Figure 2E).
  • CPTl l also had a significant immunosuppressive effect on an immune-based therapy that involves adoptive transfer of normal immune cells (donor cells).
  • donor cells normal immune cells
  • expression levels of CD247 were significantly reduced in Normal T cells that were administered to an environment which is characterized by chronic inflammation within 24 hours from the administration and upon treatment with CPT11 induced an even stronger suppressive environment, as indicated by a significant further decrease of the expression level of CD247.
  • these effects were demonstrated not only in the hosts' but also in the donor T cells.
  • Cyclophosphamide also known as cytophosphane
  • Cyclophosphamide is a chemotherapeutic drug mainly used in the treatment of the cancer types leukemia, lymphoma, breast cancer and neuroblastoma.
  • RA rheumatoid arthritis
  • this drug also decreases the levels of regulatory T cells (Treg cells), an effect which is a beneficial to the host since it decreases Treg-mediated suppression of the immune system.
  • mice in the course of chronic inflammation which are at a state of immunosuppression as observed during tumor growth, were exposed to cyclophosphamide treatment, i.p.
  • treatment with cyclophosphamide induced a decrease in the levels of Treg cells (Fig. 5A).
  • Figure 5B the levels of MDSCs in the spleen remained high, consistent with the observation during chronic inflammation.
  • Figure 5C the levels of MDSCs in the blood were significantly increased, when compared to the chronic inflammatory state.
  • chemo therapeutic agents CPT11 and cyclophosphamide are compounds that are included in a new group of drugs that act in an antagonistic manner to the immune system.
  • This group includes drugs that negatively affect the hosts' immune status and increase or maintain the chronic inflammation- induced immunosuppression.
  • the effect of treatment by these agents may be sensed by CD247, and thus CD247 may serve as a biomarker for sensing the effect of a specific chemotherapeutic agent on the immune status of the treated patient.
  • CD247 is a bio marker sensing the effect of chemotherapeutic drugs that are agonistic to the hosts' immune system
  • chemotherapeutic drugs are considered as "agonistic" to the hosts' immune system, i.e., these drugs do not enhance the immunosuppressive state of the patient.
  • the inventors therefore next examined the feasibility of using CD247 as a biomarker for sensing the beneficial effect of different "agonistic" chemotherapeutic agents on the immune status of the treated patient.
  • Doxorubicin (also known as Rubex) is a chemotherapeutic drug mainly used in the treatment of the cancer types bladder cancer, breast cancer, multiple myeloma, prostate cancer, thymoma, and others.
  • doxorubicin doxorubicin on the immune status mice in the course of chronic inflammation, which are characterized by immunosuppression as observed during tumor growth, were exposed to Rubex treatment, i.p.
  • the severity of the immunosuppressive environment was dramatically decreased, as indicated by a significant decline in MDSCs in the spleen (Fig. 8A) and in the blood (Fig.8B), while the levels of Treg cells were not affected (Fig. 8C).
  • Busulfan is a chemotherapeutic drug mainly used in the treatment of chronic myelogenous leukemia (CML) and is sometimes used for acute leukemias and lymphomas. Interestingly, it is also used in bone marrow and stem cell transplantations.
  • CML chronic myelogenous leukemia
  • Busulfan is also used in bone marrow and stem cell transplantations.
  • mice in the course of chronic inflammation which are characterized by immunosuppression as observed during tumor growth, were exposed to busulfan treatment, i.p.
  • the treatment with busulfan dramatically decreased the severity of the immunosuppressive environment, as indicated by a significant decline in MDSCs in the spleen (Fig. 10A) and in the blood (Fig. 10B), while the levels of Treg cells remained unchanged (Fig. IOC).
  • the immune status could be detected by analyzing the expression levels of CD247 in cells obtained from the spleen ( Figure 11 A), which is a further evidence for the reliability of the CD247 as a marker for the immune status; initial recovery of CD247 expression upon treatment with busulfan treatment (Figure 11 A) was observed while the levels of CD3s remained uniform among the different groups ( Figure 11B).
  • 5-fluorouracil is a chemotherapeutic drug that is mainly used in the treatment of the following types of cancers: colorectal cancer [27], basal cell carcinoma and pancreatic cancer.
  • colorectal cancer [27] colorectal cancer [27]
  • basal cell carcinoma basal cell carcinoma
  • pancreatic cancer pancreatic cancer
  • CD247 was not only capable of sensing the recovery from immunosuppression in T cells (Figure 13 A), but also in natural killer (NK) cells (Fig. 13C) and this phenomenon was associated with a recovery of the killing activity primarily mediated via NK cells ( Figure 13D and 13E).
  • the ability of the expression levels of CD247 to indicate the immune status of cells treated with 5-FU was also examined in the presence of a combined treatment that included 5-FU and immunotherapy, specifically, adoptive cell transfer.
  • a clear significant increase in the expression levels of CD247 was observed for both the host's and donor's cells, upon 5 FU treatment; the expression levels of CD247 was down-regulated in cells transferred to an inflamed host within 24 hours, as in the host cells, while in inflamed mice treated with 5-FU, the adoptive cell transfer was successful since the adoptively transferred cells showed normal CD247 expression levels as the recovered host cells.
  • CD247 may indeed serve as a reliable biomarker for detecting recovery from immunosuppression caused by a chemotherapeutic treatment, such as 5- FU or a combination with immunotherapy could be successful as long as CD247 expression levels are steady and close to normal.
  • CD247 is a bio marker sensing the effect of immune based treatment on the hosts' immune status.
  • TNFa plays a key role in the chronic inflammation induced immunosuppression, by arresting MDSCs at an immature differentiation state and increasing their suppressive activity (Sade-Feldman et. al. submitted).
  • the TNFa antagonist, etanercept was used and administrated daily, starting from 1 day before the second BCG injection, when the onset of MDSC accumulation was observed in the blood, and until 1 day before the mice were sacrificed. This regimen is schematically illustrated in Figure 15 A.
  • adoptively transferred T- and NK-cells being a treatment that is frequently used today in different immunotherapeutic regiments for various pathologies [32, 33].
  • CFSE- labeled normal splenocytes were administered, i.v., 1 day before cell harvest, at the peak of the inflammatory response (mimicking cell mediated therapies).
  • etanercept was also evaluated in-vivo, on NK-cell function under chronic inflammatory conditions. As demonstrated in Figure 15H, a reconstitution of NK cell cytotoxic activity was observed; a significant elevation in the percentage of specific allogeneic cell clearance was observed in the etanercept-treated group compared with that of the etanercept untreated group, both in the spleen and blood.
  • results presented in this Example demonstrate that treatment by etanercept enables MDSC maturation and thereby rescues the endogenous (host) immune system as well as of the newly administered (donor) cells, as manifested in the increase in the expression levels of CD247. Moreover, these results demonstrate the feasibility of using CD247 as a reliable marker for evaluating the efficacy of an immunotherapeutic treatment.
  • mice were treated with various chemotherapeutic drugs and two days after the third BCG injection mice were sacrificed and splenocytes and peripheral blood lymphocytes (PBLs) were analyzed.
  • PBLs peripheral blood lymphocytes
  • the inflammatory S 100 proteins which serve as an autocrine feedback loop, are of the key molecules controlling MDSC accumulation and retention in their immature, suppressive state induced by a given tumor, and a prolonged chronic inflammation [34].
  • chemotherapeutic drugs affect MDSC levels via this pathway.
  • Figure 17 mRNA levels of S100A8/9 (left panels) and protein levels of the S100A9 protein (right panels) were analyzed in purified MDSCs ( Figure 17 A) and in a total splenic population (Figure 17B).
  • Figure 18A and 18B show that a significant recovery of CD247 expression was observed both in the spleen and in PBLs following 5-FU treatment, as compared to its expression in chronically inflamed mice which were not administered with a chemotherapeutic treatment.
  • Figure 18 A also shows that a strong down-regulation of CD247 was observed in the spleen, with a tendency of decreased expression in PBLs following CPT-11 treatment, as compared to the expression in inflamed mice, indicating a greater suppression of CD247 in spleen with high capacity of the suppressive MDSC population.
  • NK-cells In order to investigate other effector immune cells, the impact of 5-FU and CPT-11 was also tested on NK-cells. As shown in Figure 19A, the expression of CD247 was down- regulated in NK cells obtained from chronically inflamed mice, and was found to be almost completely recovered after 5-FU treatment, whereas after the CPT-11 treatment the expression remained down-regulated with no further decrease. The in vivo activity of NK cells to eliminate transferred allogeneic cells was then investigated. To this end, splenocytes derived from syngeneic (C57BL/6) and allogeneic (BALB/c) normal mice were labeled with low and high concentrations of CFSE, respectively, and administered into C57BL/6 mice.
  • C57BL/6 syngeneic
  • BALB/c allogeneic
  • the combination may also affect the cytotoxic abilities of the drugs.
  • 5-FU inhibits DNA synthesis, which is required for the active form derivate of CPT-11 (SN38)
  • SN38 causes cell accumulation in a G2 phase, which is resistant to 5-FU [42].
  • NK cells in inflamed mice treated with the combined therapy showed less killing activity of allogeneic cells as compared with the untreated inflamed mice (Fig. 20E, 20F).
  • CPT11 treatment is able to overcome the beneficial effects of 5FU.
  • Fig. 20G when lower doses of combined chemotherapeutic agents (5FU and CPT11) were administered, the marked reduction of %MDSCs (Fig. 20G) was also followed by elevated levels of CD247 (Fig. 20H).
  • the levels of NO were also significantly reduced in response to the combined treatment (Fig. 201), but monotherapy using 5FU, showed better results also in reducing hROS (Fig. 20J).
  • mice were subjected to two injections of the mutagen azoxymethan (AOM), followed by two weekly administrations of dextran sulfate sodium (DSS), which was added to the drinking water.
  • AOM mutagen azoxymethan
  • DSS dextran sulfate sodium
  • CD247 expression levels Upon treatment with the chemotherapeutic drug 5FU, CD247 expression levels significantly recovered, while following treatment with CPT11, CD247 levels were low as in the case of CRC-bearing, non-treated mice ( Figure 21B). These results indicate that CD247 expression levels can predict the inflammatory stage and associated hosts' immune status in the course of colon cancer (CRC). Moreover, measurements of the expression levels of CD247 may also provide an indication of therapy efficacy as shown herein, upon using 5FU and CPT11 , each of them exhibiting an opposing effect on the host's immune system; while 5FU leads to a recovery from MDSC-mediated immunosuppression, CPT11 treatment maintains the immunosuppressive environment and in some cases, leads to its enhancement.
  • the immunosuppressive environment is detected within the tumor target site
  • CD247 may be used as a sensitive marker using samples obtained from the tumor tissue. Moreover, these results indicate that the levels of CD247 in the tumor tissue may predict the responsiveness of a subject to a certain treatment and therefore may be used as a prognostic tool for assessing disease progression and survival rate.
  • CD247 as a biomarker for assessing the suitability of a patient to a therapy
  • the parameters tested in the mouse model system are to be applied to the human samples (namely, the levels of MDSCs, the levels of ROS and NO production as well as the level of expression of CD247), to provide a preliminary proof of concept of using the level of expression of CD247 as a key indication to the patient's immune status prior to or during treatment, and in order to examine whether this marker may be used for staging the disease and for determining the efficacy of a given therapy on the course of the treatment.
  • This approach will enable the design of a personalized treatment, which uses CD247 as a powerful diagnostic tool.

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Abstract

L'invention concerne des méthodes, des nécessaires et des compositions utilisant la protéine CD247 en tant que biomarqueur afin d'évaluer l'efficacité d'une thérapie, par exemple d'une thérapie chimiothérapeutique, biologique ou combinée, et de sélectionner une thérapie appropriée en vue du traitement d'un sujet souffrant d'une affection pathologique conduisant à une affection inflammatoire chronique.
PCT/IL2012/050393 2011-10-04 2012-09-27 Protéine cd247 utilisée comme biomarqueur en vue de l'évaluation de l'effet de médicaments chimiothérapeutiques et biologiques WO2013050998A1 (fr)

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US14/348,621 US10955415B2 (en) 2011-10-04 2012-09-27 CD247 as a biomarker for assessing the effect of chemotherapeutic and biological drugs
EP12784738.2A EP2834640B1 (fr) 2011-10-04 2012-09-27 Protéine cd247 utilisée comme biomarqueur en vue de l'évaluation de l'effet de médicaments chimiothérapeutiques et biologiques

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